Assessment of Phagocyte Functions by Flow UNIT 9.

19
Cytometry
Phagocytes are crucial to effective defense against infections. Neutrophilic polymor-
phonuclear leukocytes (PMNs), and monocytes and macrophages (MΦs), are crucial for
cellular defense against infections. Phagocyte functions encompass phagocytosis (attach-
ment and internalization), intra- and extracellular digestion of targets, oxidative burst, and
chemotaxis. Accurate measurement of phagocytosis in one cell population requires
discrimination from other populations by light scatter or fluorescent staining. Phagocy-
tosis of most microorganisms requires serum factors (opsonins). The major opsonins are
antibodies, complement factors, and fibronectin. Flow cytometry is applied to both routine
and experimental quantification of phagocyte functions as well as to the quantitation of
opsonin and antigen concentrations and activities. Bacteria, zymosan particles, and
microspheres are used as targets for different experimental and clinical purposes. The
basic protocols in this unit outline a four-step tandem procedure for evaluating all the
above-mentioned phagocyte functions: attachment and internalization (see Basic Protocol
1), processing of bacteria and zymosan particles (see Basic Protocol 2), phagocytosis and
oxidative burst (see Basic Protocol 3), and chemotaxis (see Basic Protocol 4).

PHAGOCYTOSIS (ATTACHMENT AND INTERNALIZATION) AND BASIC

PHAGOSOMAL pH PROTOCOL 1
This protocol is a two-step procedure for measurement of total phagocytosis (attachment
and internalization) and phagosomal pH. FITC-stained bacteria and zymosan particles
are used as targets, which are mixed with opsonins and leukocytes under controlled mixing
and temperature conditions. The first step concerns measurements of percentage phago-
cytosis, phagocyte fluorescence, and the loss of targets from the suspension, followed by
calculation of the phagocytic index.
In the second step, trypan blue is added after phagocytosis, and percentage phagocytosis
and phagocyte fluorescence are recorded. The numbers of attached and internalized
targets are calculated from the phagocytic index, the fluorescence of the phagocytes
before and after trypan blue quenching, and the fluorescence of targets. The difference in
the fluorescence between the internalized and the free, extracellular targets is taken as an
indirect measure of phagosomal pH using a standard curve of pH versus FITC fluores-
cence. The intensity of the phagocyte FITC fluorescence depends on complex intracellular
mechanisms that have to be taken into account when evaluating phagosomal pH (see
Commentary).
Materials
Targets:
Bacteria S. aureus Cowan III (NCTC 8532; see Support Protocol 4)
Zymosan particles (see Support Protocol 3 and/or 5)
Polychromatic red fluorescence (PC red)-dyed microspheres, 1-µm diameter
(Fluoresbrite Plain Microspheres, Polysciences) coated with outer membrane
vesicles from meningococci (OMV-beads; see Support Protocol 7)
Sørnes’s buffer (see recipe)
Opsonins (see Support Protocol 9)
Dihydrorhodamine 123 (DHR; see recipe)
White blood cell suspension (see Support Protocol 1)
Dulbecco’s phosphate-buffered saline (DPBS; see recipe) containing 0.02% (w/v)
EDTA, ice cold Studies of Cell
Function
Contributed by Carl-Fredrik Bassøe 9.19.1
Current Protocols in Cytometry (2002) 9.19.1-9.19.22
Copyright © 2002 by John Wiley & Sons, Inc. Supplement 21
 Trypan blue
Vindeløv’s high-salt solution (see recipe)
Fluorescent beads (e.g., DNA-Check, Coulter)
Flow cytometer with 488-nm excitation and filters for the detection of green
fluorescence (R123, 505 to 545 nm), orange-red fluorescence (PC red beads,
560 to 590 nm), and red fluorescence (CD14-PE-Cy5, 660 to 700 nm)
Non-pyrogenic 96-well microtiter plates, sterile
12 × 75–mm tubes suitable for use on the flow cytometer

Prepare targets
1. Thaw selected targets. Calculate target concentration by flow cytometry using a
known target volume (VT), and a known volume (VL) and concentration (CL) of
leukocytes as internal reference, and note percent leukocytes (PL) and percent targets
(PT).
Target concentration = (PT/PL) × (CLVL/VT).
Targets are prepared and kept for several years. See specific support protocols for target
preparation.
2. Adjust bacteria and zymosan targets to 2.5 × 108 and 1.25 × 108 per milliliter in
Sørnes’s buffer, respectively.
Targets are adjusted to a useful concentration relative to the amount of added FITC, and
the applied quenching. The initial target/phagocyte (PMN + MΦ) ratio (R) is 20:1 for
bacteria and 10:1 for zymosan particles.

Incubate cells
3. Add 20 µl each of targets, diluted opsonins, and DHR working solution to each well
of 96-well microtiter plates, and adjust the final incubation mixture to 80 µl per well
by adding 20 µl Sørnes’s buffer. Use an additional duplicate plate (termed QUICK-
MIX) for controls. Add all the mentioned ingredients, except diluted opsonins, to the
QUICK-MIX plate.
4. Incubate 7.5 min at 37°C.
5. Add 20 µl white blood cell suspension to each well.
6. Incubate suspensions 7.5 min for phagocytosis, and 30 min for processing (see Basic
Protocol 2) under controlled mixing at 37°C.
7. Stop mixing, and immediately add 200 µl ice-cold DPBS containing 0.02% EDTA
to each well using the same sequence of additions as for the cells in order to equalize
phagocytosis time for all the wells. Add 20 µl diluted opsonins to the QUICK-MIX
plate.
8. Label three 12 × 75–mm tubes for each well. Add 400 µl DPBS with 0.02% EDTA
to one set, 400 µl of 2 mg/ml trypan blue in DPBS with 0.02% EDTA to the second
set, and 400 µl Vindeløv’s high salt-solution to the third set.
The first set of tubes is used for determination of percentage phagocytosis, phagocyte
fluorescence, target loss, and phagocytic index. The second set of tubes is used for
discrimination between attachment and internalization. The third set of tubes is reserved
for processing in Basic Protocol 2.
9. Mix the contents of each well in the 96-well plate thoroughly using a pipet (cells and
Assessment of targets sediment at different rates). For each well, transfer 100 µl suspension to each
Phagocyte of the three labeled tubes (step 8) corresponding to that well. Keep the tubes on ice
Functions by
Flow Cytometry and in the dark until flow cytometry measurement.
9.19.2
Supplement 21 Current Protocols in Cytometry
 A
1023
L

Forward scatter

0
0 1023
Side scatter

B
1023 N1 N2
Forward scatter

N3 N4

0
100 101 102 103
FITC fluorescence

C
1023 O1 O2
Forward scatter

O3 O4

0
100 101 102 103
FITC fluorescence

Figure 9.19.1 The monocytes (M) and neutrophils (L) in the FS versus SS cytogram of leukocytes
phagocytosing bacteria or zymosan particles are gated to make separate FS versus FITC
fluorescence cytograms of monocytes and neutrophils (A). Percentage statistics and mean FITC
fluorescence values are recorded in the latter cytograms (B). The third cytogram (C) of FS versus
FITC fluorescence gives the relative counts of phagocytes and free targets. The phagocytic index
is calculated from the latter relative counts. Studies of Cell
Function

9.19.3
Current Protocols in Cytometry Supplement 21
 10. Mix the contents of each tube thoroughly (cells and targets sediment at different rates)
just prior to running on the flow cytometer.

Acquire data
11. Calibrate flow cytometer with fluorescent beads (e.g., DNA-Check, Coulter). Use
electronic color compensation (UNIT 1.14) to minimize the spectral emission overlaps
between fluorochromes.
Coincidence rate of targets and leukocytes in the flow cell should be 1% to 2% (<5%).
12. Run the first set of tubes (DPBS with 0.02% EDTA only). Collect forward scatter
(FS), side scatter (SS), and yellow-green FITC fluorescence (FITC; Fig. 9.19.1).
Create ungated FS versus FITC cytogram (total cytogram).
13. Set region about noise. For the phagocytosis cytogram, make gated FS versus FITC
from cytogram by subtracting the noise region to create phagocytosis cytogram.
14. Create quadrant statistics in phagocytosis cytogram. Place the vertical limit close to
the left of the targets. Place the horizontal close beneath the lymphocytes. Record
quadrant statistics: region 1 contains non-phagocytes (N), region 2 contains phago-
cytes (F), and region 4 contains targets (T).
15. Record percentage phagocytosis:
P% = 100 × F%/(F% + N%).
16. Calculate average phagocytic index:
IP = R – (T%/F%).
R is the initial target/phagocyte (PMN + MΦ) ratio.
17. Note the mean fluorescence of the free, extracellular targets (FTe).

Create PMN and MΦ cytograms

18. Make gated FS versus FITC cytograms by gating on PMN and MΦ, respectively, in
the FS versus SS cytogram. Create quadrant statistics by placing the vertical and
horizontal limits as described in step 14.
19. Record quadrant statistics: region 1 contains non-phagocytosing PMNs or MΦs (N);
region 2 contains phagocytosing PMNs or MΦs (F).
20. Calculate PMN percentage phagocytosis:
PCPMN% = 100 × FCPMN%/(FCPMN% + NCPMN%).
21. Calculate MΦ percentage phagocytosis:
PCMΦ% = 100 × FCMΦ%/(FCMΦ% + NCMΦ%).
22. Record the mean fluorescence of the phagocytosing PMNs (FCPMN) and the mean
fluorescence of the phagocytosing MΦs (FCMΦ).

Determine attachment and internalization

23. Run the second set of tubes (trypan blue–containing samples) with the same flow
cytometer settings and record PMN percentage phagocytosis (PQPMN%).
24. Calculate percentage of PMNs with only adherent targets:

Assessment of PAPMN% = PCPMN% – PQPMN%.

Phagocyte 25. Record MΦ percentage phagocytosis (PQMΦ%).
Functions by
Flow Cytometry

9.19.4
Supplement 21 Current Protocols in Cytometry
26. Calculate percentage of MΦs with only adherent targets:
PAMΦ% = PCMΦ% – PQMΦ%.
27. Record the mean fluorescence of the phagocytosing PMN (FQPMN) and the mean
fluorescence of the phagocytosing MΦs (FQMΦ).
28. Calculate the mean number of targets attached to each PMN:
IAPMN = (FCPMN – FQPMN)/FTe.
29. Calculate the mean number of targets attached to each MΦ:
IAMΦ = (FCMΦ – FQMΦ)/FTe.
30. Calculate the mean fluorescence of the targets internalized by the PMNs:
FTiPMN = FQPMN/(IPMN × FTe).
31. Read the phagosomal pH off the pH versus fluorescence standard curve. Establish a
standard curve by measuring target fluorescence in suspensions with pH 4 to 10 at
0.5-pH step intervals.

PROCESSING OF BACTERIA AND ZYMOSAN PARTICLES BASIC

PROTOCOL 2
Phagocytes are added to Vindeløv’s high-salt solution containing the detergent NP40. In
a single step, phagocytes are lysed, most of the targets are liberated, and some adhere to

1000
digestion

100 Nc Nc − Z
EB fluorescence

10

.1
.1 1 10 100 1000
FITC fluorescence

Figure 9.19.2 Cytogram for the measurement of digestion. Cytogram of phagocytosing leuko-
cytes after the dissolution of the leukocyte cytoplasm in Vindeløv’s solution. Region Nc (EB-fluores-
cent nuclei) contains mainly lymphocyte nuclei. Region Nc + Z (EB- and FITC-fluorescent nuclei)
contains monocyte and neutrophil nuclei with 1 to 3 attached targets. Region Z contains free targets
(bacteria or zymosan particles). The mean EB and FITC fluorescence values are recorded from
Studies of Cell
region Z. Function

9.19.5
Current Protocols in Cytometry Supplement 21
 the cell nuclei. Target DNA is stained with ethidium bromide (EB), and RNA is dissolved
by RNase. Flow cytometry measurements on FS, SS, and FITC and EB fluorescence can
be made on individual leukocyte nuclei and leukocyte nuclei with a few adherent targets,
as well as on the liberated, free targets and those that remained extracellularly during
phagocytosis.

Materials
Cells in Vindeløv’s high-salt solution from Basic Protocol 1, step 9
Flow cytometer with 488-nm excitation and filter set for detection of green (FITC)
and red (EB) fluorescence

A
103
CD14-PE-Cy5 fluorescence

102

101

A
100

0 1023
Side scatter

B
103
D1D2

102
R123 fluorescence

101

100

D3D4
100 101 102 103
PE-labeled OMV-beads

Figure 9.19.3 After the phagocytosis of OMV-beads, monocytes and neutrophils cannot be
discriminated by FS versus SS cytogram. Therefore, monocytes and neutrophils are stained with
PE-Cy5-labeled antiCD14 monoclonal antibody, and discriminated by PE-Cy5 fluorescence and
SS (A). Monocytes are positive for CD14 and are in region B, whereas, neutrophils are negative or
weakly CD14-positive, and located in region A. Monocytes and neutrophils are gated to separate
Assessment of cytograms (B) for measurement of phagocytosis (PE-fluorescence of OMV-beads) and oxidative
Phagocyte
Functions by burst (R123 fluorescence). The mean PE and R123 fluorescence of double-positive cells are
Flow Cytometry recorded from region D2, and that of single-positive cells from regions D1 and D4.

9.19.6
Supplement 21 Current Protocols in Cytometry
Acquire data

For bacteria targets

1a. Collect yellow-green FITC fluorescence and red EB fluorescence, and make log FITC
versus log EB cytogram. Place electronic gating region about individual cell nuclei
(Fig. 9.19.2, region Nc), cell nuclei with adherent targets (Fig. 9.19.2, region Nc +
Z), and free and liberated target cell nuclei (Fig. 9.19.2, region Z).
2a. Record mean fluorescence values on region Z in Figure 9.19.2.
3a. Compare mean green FITC and red EB fluorescence of QUICK-MIX with that of
PHS and PHS-IN.

For zymosan particles

1b. Make appropriate regions and collect information as in steps 1a to 3a (new settings
are required due to the higher FITC and EB fluorescence intensities of the zymosan
particles).
2b. Gate on region Z in Figure 9.19.2 into FS versus SS cytogram.
3b. Record mean FS and SS from the targets, and compare mean FS and SS of QUICK-
MIX (see below) with that of HYP and HYP-IN.

PHAGOCYTOSIS AND OXIDATIVE BURST BASIC

PROTOCOL 3
This protocol is used for the concurrent measurement of phagocytosis and oxidative burst.
Various microspheres can be used as targets (see Commentary). In this case, phagocytosis
is monitored by polychromatic red fluorescent (PC red) microspheres coated with outer
membrane vesicles from meningococci (OMV-beads). The oxidative burst is quantified
by the conversion of dihydrorhodamine123 (DHR) to green fluorescent rhodamine 123
(R123). PMNs and MΦs are discriminated using an orange fluorescent monoclonal
antiCD14 antibody. Data are simultaneously collected on targets, PMNs, and MΦs.
Additional Materials (also see Basic Protocol 1)
CD14-PE-Cy5 monoclonal antibody
Desired microspheres (see Support Protocol 7), adjusted to a concentration of 2.5
× 108 /ml in Sørnes’s buffer (see recipe); the initial microsphere/phagocyte
(PMN + MΦ) ratio (R) is 20:1.

Incubate cells
1. Follow Basic Protocol 1, steps 3 to 7.
2. Prepare microtiter plates in advance by adding 5 µl undiluted monoclonal antibody
to the desired number of wells. Add 100 µl cell suspension to each well, mix the
suspension gently, and incubate 1 hr in the dark on ice.
3. Mix the contents of each well thoroughly using a pipet and transfer to a 12 × 75–mm
tube containing 400 µl DPBS with 0.02% EDTA and proceed with flow cytometry.

Acquire data
4. Create cytogram of CD14 versus SS. Set separate regions about noise and small
particles, monocytes, and neutrophils. Also create a histogram on red fluorescence
(targets and phagocytes) gated from debris and small particles. Collect statistics on
free targets, and record mean target red fluorescence.

Studies of Cell
Function

9.19.7
Current Protocols in Cytometry Supplement 21
 5. Create two cytograms to monitor phagocytosis and oxidative burst by monocytes and
neutrophils by placing quadrant statistics as described in Basic Protocol 1 (Fig.
9.19.3). Record the percentages in all regions.
6. Percentage phagocytosis P% = percentage of counts in region 2 plus region 4.
Percentage oxidative burst PO% = percentage of counts in region 1 plus region 2.
Percentage of inactive cells = percentage from region 3. Percentage of non-phagocy-
tosing cells producing oxidative burst = percentage from region 1. Percentage of
phagocytosing cells without oxidative burst = percentage from region 4.
7. Record the mean green fluorescence in region 1 (oxidative burst alone), green and
red fluorescence in region 2 (oxidative burst and phagocytosis), and red fluorescence
in region 4 (phagocytosis only).
8. For region 2 in cytograms from both monocytes and neutrophils, calculate phagocytic
index IPMN (IMΦ) = mean red fluorescence of phagocytes in region 2 divided by the
red fluorescence of the free targets.
9. For region 2, calculate neutrophil oxidative ratio OPMN = mean green fluorescence of
phagocytes in region 2 divided by IPMN, and monocyte oxidative ratio OMΦ = mean
green fluorescence of phagocytes in region 2 divided by IMΦ.

BASIC CHEMOTAXIS
PROTOCOL 4
Measurement of chemotaxis is based on the selective, active, and stimulated motion of
neutrophils across a 3-µm filter. The source population serves as a reference, and
non-stimulated cells as a control. Two or more chemotactic stimuli, zymosan-activated
serum (ZAS) and formyl-methionyl-leucyl-phenylalanine (fMLP), are routinely used.
Materials
White blood cell suspension (see Support Protocol 1)
Sørnes’s buffer (see recipe)
DPBS (see recipe) containing 0.2% (w/v) EDTA
5% zymosan-activated serum (ZAS; see Support Protocol 3)
Paraformaldehyde (optional)
12 × 75–mm tubes
24-well transwell chemotaxis plates (6.5-mm diameter, 3-µm pore size; Corning)
37°C, 5% CO2 incubator

Prepare cells
1. Centrifuge white blood cell suspension 5 min at 350 × g, 20°C, and resuspend in
Sørnes’s buffer to a final concentration of 5 × 106 leukocytes/ml.
2. For reference sample, add 2 ml DPBS containing 0.2% EDTA, 100 µl cell suspension,
and 500 µl Sørnes’s buffer to each of two 12 × 75–mm tubes.
These tubes contain the same volume as the final dilution in the lower well, and are the
100% cell count.
3. Add 600 µl Sørnes’s buffer to the lower chamber of two wells of the 24-well transwell
microtiter plate (control).
4. Add 600 µl of 5% ZAS (or other stimulus) in Sørnes’s buffer to the lower chamber
of two wells of the 24-well transwell microtiter plate (control).
Assessment of 5. Add 100 µl cell suspension (5 × 106 leukocytes/ml) to the corresponding upper
Phagocyte chambers of the 24-well transwell microtiter plate.
Functions by
Flow Cytometry

9.19.8
Supplement 21 Current Protocols in Cytometry
 6. Incubate 30 min in a 37°C, 5% CO2 incubator. After the incubation, immediately
wash upper and lower chambers each with 2 ml ice-cold DPBS containing 0.2%
EDTA, and transfer the leukocytes to 12 × 75–mm tubes. Keep the cell suspensions
on ice and proceed with flow cytometry, or fix cells in 1.0% final paraformaldehyde
for later investigation.

Acquire data
7. Record total and differential counts of lymphocytes, MΦs, and PMNs using FS
versus SS and appropriate gates from reference sample and the lower chamber.
8. Calculate spontaneous (random) motion = 100 × PMN count in lower chamber
without stimulus divided by PMN count in reference suspension.
9. Calculate chemotaxis = 100 × PMN count in lower chamber with stimulus divided
by PMN count in reference suspension.

PREPARATION OF WHITE BLOOD CELL SUSPENSION SUPPORT

PROTOCOL 1
This protocol describes the isolation of a white blood cell suspension from peripheral
blood. The suspension provides starting material for the basic protocols.

Materials
Lysing solution (see recipe)
Dulbecco’s phosphate buffered saline with glucose and BSA (DPBS-GA; see
recipe)
Vacutainer tubes containing 100 U preservative-free heparin (UNIT 9.7)
50-ml centrifuge tubes, sterile
Additional materials for blood collection
1. Collect peripheral blood sample in the Vacutainer tube containing heparin.
2. In a 50-ml centrifuge tube, dilute heparinized blood 1:10 (v/v) in lysing solution, mix
gently, and leave 10 min at room temperature.
3. Centrifuge 5 min at 350 × g, room temperature, resuspend the pellet in 20 ml washing
solution, and centrifuge again 5 min at 350 × g, room temperature.
4. If lysis is incomplete, resuspend the pellet 5 min in NH4Cl solution (final 8 mg/ml),
pH 7.4, centrifuge 5 min at 350 × g, room temperature, and resuspend in 20 ml
DPBS-GA.
5. Centrifuge 5 min at 350 × g, room temperature, and resuspend pellet in 1 ml
DPBS-GA per 10 ml of starting heparinized blood.
6. Adjust the concentration to 1.25 × 107 white cells/ml in DPBS-GA.

Studies of Cell
Function

9.19.9
Current Protocols in Cytometry Supplement 21
 SUPPORT PREPARATION OF SERUM FROM PERIPHERAL BLOOD
PROTOCOL 2 Blood is collected in serum tubes with no anticoagulant and is allowed to stand undis-
turbed until it clots. The clot is removed and the remaining liquid is centrifuged to remove
any residual erythrocytes and debris. The resulting serum is divided into aliquots and
frozen.
Materials
Freshly extracted peripheral blood
Red-top collection tubes (no anticoagulant)
50-ml centrifuge tubes
Additional equipment for venipuncture
1. Place tube of blood in a rack and allow to sit undisturbed until a clot forms.
Blood must be collected without an anticoagulant.
If a clot does not start to form within 15 min, initiate clotting by inserting a wooden
applicator stick into the tube. Then begin with step 1.
2. Using a wooden applicator stick, gently loosen the clot and remove from the tube.
Do not break up clot.
3. Transfer serum to 50-ml centrifuge tube and centrifuge 10 min at 2700 × g, 4°C.
4. Save the supernatant.
5. Combine the supernatants from at least 7 different donors, divide into 500-µl aliquots
or other desired volume, and store in appropriate containers at –20°C.
Do not subject sera to repeated freeze-thaw cycles.

SUPPORT ZYMOSAN-ACTIVATED SERUM (ZAS) AND PREOPSONIZED ZYMOSAN

PROTOCOL 3 PARTICLES
ZAS is used as one stimulus for cell motility in the chemotaxis assay. Preopsonized
zymosan particles are used in the assay for complement-mediated phagocytosis. ZAS and
complement fragment C3b- and C3bi-opsonized zymosan particles are prepared simul-
taneously.
Materials
Zymosan A particles (Sigma)
DPBS (see recipe)
Fresh human serum (see Support Protocol 2)
Sørnes’s buffer (see recipe)
15-ml plastic centrifuge tube
37°C incubator with rotator
1. Add 100 mg zymosan A particles and then 10 ml DPBS to a 15-ml plastic centrifuge
tube and shake vigorously.
2. Centrifuge 10 min at 2000 × g, 20°C.
3. Pour off supernatant and add 12.5 ml fresh human serum. Shake and rotate solution
1 hr at 37°C.
4. Centrifuge 10 min at 2000 × g, 20°C, decant serum and label as ZAS, and store up
to 5 years at –80°C.
Assessment of 5. Resuspend zymosan A pellet in 10 ml Sørnes’s buffer. Use immediately or store as
Phagocyte
Functions by preopsonized zymosan particles in 1-ml aliquots up to 5 years at –80°C.
Flow Cytometry

9.19.10
Supplement 21 Current Protocols in Cytometry
FITC-LABELING OF BACTERIA SUPPORT
PROTOCOL 4
FITC-labeled bacteria are used to investigate adhesion of bacteria to leukocytes, and
antibody-dependent and antibody-plus-complement-stimulated phagocytosis.
Materials
Fluorescein isothiocyanate (FITC)
96% and 70% ethanol
Heart infusion broth (HIB; Difco)
S. aureus Cowan III (NCTC 8532 O/N)
Blood agar plates (e.g., Becton Dickinson)
0.9% (w/v) NaCl
Sørnes’s buffer (see recipe)
Spectrophotometer
1. Weigh out 10 mg FITC, and add to 0.1 ml of 96% ethanol. Dilute the FITC-ethanol
solution to 100 ml in heart infusion broth (HIB).
2. Grow S. aureus Cowan III on blood agar, and add bacteria to ∼2 ml HIB. Mix until
the suspension is free from clumps. Warm 100 ml HIB to 37°C and add the 2-ml S.
aureus suspension. Read the OD using a spectrophotometer (the OD of the suspension
should be 0.05 to 0.1 at 620 nm). Incubate at 37°C until OD = 1.0 (∼2.5 hr).
3. Pellet the bacteria by centrifuging 10 min at 2000 × g, room temperature. Resuspend
bacteria in ∼2 ml of 0.9% NaCl. Mix until there are no visible bacteria clumps.
4. Dilute bacteria in ∼90 ml ice-cold 70% ethanol to OD=1.0. Let the bacteria fix 30
min at 0°C. Repeat step 3 three times at 4°C.
5. Dilute the bacteria in two 50-ml tubes each containing 30 ml FITC solution (see step
1). Incubate 30 min at 37°C with continuous mixing.
6. Repeat step 3 three times. Resuspend bacteria in Sørnes’s buffer, and adjust to OD =
1. Divide suspensions into 0.5-ml aliquots and store FITC-stained bacteria up to 5
years at –80°C.

FITC-LABELING OF ZYMOSAN PARTICLES SUPPORT

PROTOCOL 5
Phagocytosis is mediated by complement fragments, mainly C3b and C3bi. Zymosan
particles bind complement factor C3 that is converted into C3b and C3bi, which remain
covalently bound on the particles’ surface. FITC-labeled zymosan particles are used to
measure complement-mediated phagocytosis, and intracellular processing mediated by
complement.
Additional Materials (also see Support Protocol 4)
Zymosan A particles
DPBS (see recipe)
10-ml glass tubes
0.2-µm filter
End-over-end rotator, 37°C
1. Add 100 mg zymosan A particles to each of ten 10-ml glass tubes.
2. Make FITC solution: Place 10 mg FITC in a 10-ml glass tube. Add 0.5 ml of 96%
ethanol to dissolve the FITC. Add 9.5 ml DPBS at 37°C to a final 1 mg/ml FITC
stock solution. Dilute with DPBS at 37°C to a working solution containing 0.05 Studies of Cell
mg/ml FITC. Function

9.19.11
Current Protocols in Cytometry Supplement 21
 3. Filter sterilize through a 0.2-µm filter.
4. Add 10 ml of 0.05 mg/ml FITC solution to each glass tube with zymosan particles.
Incubate 30 min at 37°C with end-over-end rotation.
5. Centrifuge 10 min at 2000 × g, room temperature, and resuspend the pellet in DPBS.
Repeat three times.
6. Centrifuge 10 min at 2000 × g, 20°C, and resuspend the pellet in 200 ml Sørnes’s
buffer. Store 0.5-ml aliquots protected from light up to 5 years at –80°C.

SUPPORT DILUTIONS OF FITC-LABELED ZYMOSAN A PARTICLES

PROTOCOL 6
This protocol is used for the adjustment of FITC concentration, in case the FITC-stained
particles are not quenched by trypan blue.
Additional Materials (also see Support Protocol 5)
Trypan blue
15-ml polypropylene tubes
1. Add 100 mg zymosan A particles and 2 ml DPBS to a 15-ml polypropylene tube. Mix
with a pipet or ultrasound (30 sec) until the suspension is free from clumps. Dilute
to 10 ml with DPBS. Centrifuge 10 min at 2000 × g, room temperature, and discard
the supernatant.
2. Dissolve 0.25 mg FITC in 0.1 ml of 96% ethanol and immediately dilute in 1 ml
DPBS at 37°C. Wait until all FITC is solubilized. Pass through a 0.22-µm filter. Make
the following concentrations of FITC in DPBS: 0.25, 0.1, 0.05, and 0.025 mg/ml.
3. Resuspend zymosan A pellet in a little FITC solution and mix until free from clumps.
Thereafter, add 10 ml FITC solution per 100 mg zymosan particles. Incubate with
end-over-end rotation 30 min at 37°C.
4. Centrifuge 10 min at 2000 × g, room temperature. Decant and resuspend in 10 ml
room temperature 0.9% NaCl. Repeat this step three times.
5. Resuspend the pellet in 10 ml DBPS. Count zymosan particles using flow cytometry
(see Support Protocol 8) and dilute to 5 × 108 particles/ml.
6. Run suspension on flow cytometer and place the modal FITC fluorescence in channel
∼100.
7. Add 100 µl FITC-labeled zymosan suspension to 400 µl trypan blue suspension. Run
on flow cytometer. Use the solution with the highest FITC concentration that is 100%
effectively quenched.
8. Store the selected FITC-stained zymosan population in 1-ml aliquots up to 5 years
at –80°C.

Assessment of
Phagocyte
Functions by
Flow Cytometry

9.19.12
Supplement 21 Current Protocols in Cytometry
ANTIGEN COATING OF POLYSTYRENE BEADS SUPPORT
In principle, beads are incubated with an excess of antigen overnight at room temperature PROTOCOL 7
(20°C). Unreacted sites on the beads are blocked with 2% BSA. Antigen-coated beads
are suspended in a storage buffer, and kept protected from daylight at 4°C.
OMV from N. meningitidis strain 44/76 (B:15:P1.7,16) is prepared as for vaccine
production (Lehmann, 1997).
Materials
Fluoresbrite PC red 1.0-µm microspheres (Polysciences)
0.1 M borate buffer, pH 8.5 (0.1 M boric acid; Polysciences)
OMV-beads with and without 2% (w/v) bovine serum albumin, endotoxin-free
(BSA; Roche Diagnostics)
Sodium phosphate storage buffer (Polysciences)
End-over-end rotator
1. Centrifuge 500 µl Fluoresbrite PC red microspheres (4.55 × 1010 microspheres/ml)
5 min at 15,600 × g, room temperature. Resuspend pellet in 550 µl of 0.1 M borate
buffer, pH 8.5.
2. Add 450 µl of a 0.94 mg/ml (saturating) OMV suspension. Mix by end-over-end
rotation 20 hr in the dark at room temperature.
3. Centrifuge OMV-beads 5 min at 15,600 × g, room temperature, and resuspend them
in OMV suspension containing 2% endotoxin-free BSA. Incubate with end-over-end
rotation 20 hr at 20°C.
4. Centrifuge OMV-beads with blocked free surface sites 5 min at 15,600 × g, room
temperature, and resuspend in sodium phosphate storage buffer. Store up to 1 year at
4°C.

COUNTING TARGETS SUPPORT

For the quantification of small particles, leukocytes of a known concentration are used as PROTOCOL 8
reference. Mix known volumes of leukocytes and beads. Discriminate leukocytes, debris,
and fluorescent beads by forward and side scatter. Place electronic windows around
targets and leukocytes and record the relative count of beads and leukocytes. Calculate
the concentration of beads by multiplying the concentration of leukocytes by the relative
bead/leukocyte ratio.

OPSONINS SUPPORT
The phagocytosis of many pathogenic microorganisms and fungi requires serum opson- PROTOCOL 9
ins. Complement is a natural opsonin for many microorganisms and fungi. Complement
factor 3 (C3) binds to the surface of many non-capsulated microorganisms and zymosan
particles. C3 is converted to C3b and C3bi that remain attached to the targets, and bind
to complement receptors on phagocytes. For example, C3bi binds to complement receptor
3(CD11b). Hypo- and agammaglobulinemic sera are useful sources of complement. Due
to the low antibody concentration, they are used in conjunction with zymosan particles
to monitor complement-mediated phagocytosis.
Infections normally induce the synthesis of antibodies. In the acute phase (day 0 to 2),
the concentration of specific antibodies against the infectious agent is low (acute-phase
serum). After 2 to 4 weeks, the concentration of specific antibodies is high (convalescent
serum). Antibodies bind to the microorganisms by the Fab part, and link them to the
phagocytes through the Fc part. The Fc part of IgG promotes phagocytosis by binding to
Fc-receptors FcγRII (CD32) and FcγRIII (CD16) on neutrophils, and FcγRII (CD32) on Studies of Cell
Function

9.19.13
Current Protocols in Cytometry Supplement 21
 monocytes. Convalescent serum is used as a source of specific antibodies. Acute-phase
serum serves as control.

Materials
Healthy human serum
Hypogammaglobulinemic serum
Acute-phase and convalescent serum
Sørnes’s buffer (see recipe)

For a pooled serum from healthy controls

1a. Collect serum (see Support Protocol 2) from healthy individuals, and ensure that CH50
is within the normal reference range.
2a. Mix equal volumes of serum from each of seven healthy individuals and label as
pooled human serum (PHS). Heat-inactivate a portion (step 3a) and store the
remainder in 0.5-ml aliquots up to 5 years at –80°C.
3a. Inactivate complement by heating 30 min at 56°C. Label as PHS-IN and immediately
store in 0.5-ml aliquots up to 5 years at –80°C.
4a. Thaw shortly before use.

For hypogammaglobulinemic serum

1b. Collect hypogammaglobulinemic serum (HYP) from agammaglobulinemic patients
before immunoglobulin substitution. Heat-inactivate a portion (step 2b) and store the
remainder in 0.5-ml aliquots up to 5 years at –80°C.
2b. Inactivate complement by heating 30 min at 56°C. Label as HYP-IN. Immediately
after preparation store in 0.5-ml aliquots up to 5 years at –80°C.
3b. Thaw shortly before use.

For acute-phase and convalescent serum

1c. For antigen- and antibody-dependent phagocytosis: collect acute-phase serum (APS)
from patient on admission, and convalescent serum (REC), 6 weeks later. Heat-inac-
tivate a portion (step 2c) and store the remainder in 0.5-ml aliquots at –80°C.
In the example presented here, sera collected at admission and 6 weeks later from a patient
with meningococcal C:15:P1.7,16 disease are used.
2c. Inactivate complement by heating 30 min at 56°C. Label as REC-IN. Immediately
after preparation store in 0.5-ml aliquots up to 5 years at –80°C.
3c. Thaw shortly before use.
4c. Dilute 0.5 ml of each serum (APS, REC) 1:4 in Sørnes’s buffer.
OMV is obtained from serogroup B meningococci vaccination strain 44/76, B:15:P1.7.16.
OMV-suspension consists of 50 ìg protein and 3.5 ìg lipopolysaccharide per ml suspen-
sion.

Assessment of
Phagocyte
Functions by
Flow Cytometry

9.19.14
Supplement 21 Current Protocols in Cytometry
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Dihydrorhodamine (DHR) 123

Stock solution: 10 mg/ml in dimethylsulfoxide (DMSO). Store protected from light
≤3 weeks at –80°C.
Working solution: Dilute 1:1000 in Sørnes’s buffer (see recipe) before use.
Dulbecco’s phosphate buffered saline (DPBS)
Make 1 liter:
8 g NaCl
1.44 g Na2HPO4.2H2O
0.2 g KCl
0.2 g KH2PO4
Adjust to pH 7.4
Store ≤2 months at 4°C
Dulbecco’s phosphate buffered saline with glucose and BSA (DPBS-GA)
100 ml DPBS (see recipe)
0.5 g BSA
0.1 g glucose
Adjust to pH 7.4
Make fresh daily
Lysing solution
Make 1 liter:
8 g NH4Cl
0.8 g NaHCO3
0.88 g EDTA.2H2O
pH ≈ 6.8
Store ≤2 months at 4°C
Sørnes’s buffer
100 ml DPBS-GA (see recipe)
13.2 mg CaCl2.2H2O
12.1 mg MgSO4.7H2O
Adjust to pH 7.4
Make fresh daily
Vindeløv’s high-salt solution
Make 1 liter:
0.01 M glycine
0.7507 g NaOH
9.8575 mg ethidium bromide (2.5 × 10-5 M)
1 ml Nonidet P-40 (0.1%, v/v)
17.532 g NaCl (0.3 M)
pH should be 10
Store up to 1 year at 4°C.
Just before use, add 700 U/liter ribonuclease from 100 U/ml stock (see recipe).
Ribonuclease
Stock solution 1 mg/ml = 100 U/ml
Dissolve in 50 mM Tris⋅Cl, pH 8 (APPENDIX 2A). Store in 0.5-ml aliquots at –80°C.
Studies of Cell
Function

9.19.15
Current Protocols in Cytometry Supplement 21
 COMMENTARY
Background Information
General considerations
Many specific disorders of phagocyte func-
tions are known, such as chronic granuloma-
tous disease, CD11b/CD18 deficiency, lazy
leukocyte, and Chediak-Higashi syndrome.
These are hereditary syndromes that manifest
themselves in childhood. However, little is
known concerning phagocyte functions in
common, acquired clinical disorders of adults
and the elderly. Surprisingly little has been
done on leukemia, myelodysplasia, and bone
marrow failure. For example, up to the year
2000, only 14 investigations of phagocyte func-
tions in leukemia were available. Therefore, it
is difficult to advise on the selection of useful
methods in acquired immunodeficiencies re-
lated to phagocyte functions.
Several methods are available for the moni-
toring of phagocyte functions. The present unit
aims at in-depth investigations of phagocyte
functions that reflect the tandem attack and
processing of pathogenic microorganisms. Ba-
sic Protocols 1, 3, and 4 can be applied as
stand-alone procedures. Digestion cannot be
monitored without knowing that phagocytosis
has taken place to a sufficient degree to monitor
intracellular processing. Therefore, if Basic
Protocol 2 is applied, Basic Protocol 1 must be
used in conjunction.
All four basic protocols can be used together
on a routine basis. The protocols are fine-tuned
to minimize the workload. For example, 20 µl
of each reagent is added to all microwells to
minimize preparation time, and the same buffer
(DPBS) is used for cell preparations, dye and
serum dilutions, and all incubations.
All investigations are done in duplicate. Re-
producibility is excellent. Day-to-day and in-
terindividual variations with leukocytes from
healthy individuals are small.
Less laborious methods
Simpler methods are available, but these rely
on assumptions that require careful controls.
Technical details that might seem trivial are
quantitatively important. The most important
caveats are addressed below.
Time-consuming isolation of leukocytes has
been a major concern in working with flow
cytometry and phagocytosis. Whole-blood
Assessment of methods have the advantage of being the least
Phagocyte time consuming, and are useful in screening.
Functions by The effect of serum opsonins cannot be dis-
Flow Cytometry
9.19.16
Supplement 21
criminated from cellular defects. If a deviation
from the expected values is observed, it is
impossible to know whether the unexpected
results are caused by serum opsonins or leuko-
cytes. In such instances, the present methods
are needed as controls. It should also be noted
that diminished phagocyte function can be
compensated by effects of serum factors. Even
when the overall results are within the reference
range, whole-blood assays may miss important
deviations from the reference range.
Targets
The purpose of the investigation determines
the choice of target. Since sera and targets vary,
there is no universal calibration method for
phagocytosis.
Zymosan particles
In brief, zymosan particles are phagocyto-
sed by complement, mainly C3bi, and are se-
lected to monitor serum C3 opsonic activity and
complement receptor activity (mainly
CD11b/CD18). In HYP, 95% to 100% of PMNs
and MΦs phagocytose zymosan particles. With
HYP-IN, neither PMNs nor MΦs phagocytose
zymosan particles.
Two-color investigations using directly con-
jugated antibodies show that all the PMNs and
MΦs from the same suspensions carry comple-
ment receptor 1 (CR1, CD35) and complement
receptor 3 (CR3, CD11b/CD18).
Both live and dead microorganisms can be
used. S. aureus is used to monitor serum opson-
ins (mainly IgG), Fcγ-receptors, and the coop-
eration between IgG and complement. PHS and
PHS-IN are used for combined IgG-comple-
ment phagocytosis and IgG-mediated phago-
cytosis, respectively. Assessment of phagocy-
tosis using microorganisms is influenced by
properties other than antigenic properties of
microorganisms. Intracellular sorting in MΦs,
e.g., differs between various strains of the same
microbial species.
Microorganisms containing green fluores-
cent protein (GFP) are widely used for moni-
toring phagocytosis. Some technical problems
have not been addressed. For example, the kill-
ing and digestion of GFP by powerful phago-
cyte enzymes is usually not taken into account.
Caution is required with interpretation of the
results of fluorescence measurements.
Current Protocols in Cytometry
Microspheres
Coated beads have been used in phagocy-
tosis assays for a long time, but the use of
bead-associated antigens in the quantification
of phagocytosis is of recent date (Bassøe et al.,
2000). The advantage of using fluorescent
beads lies in the ability to quantify the phago-
cytic and oxidative responses in mixed cell
populations.
Microspheres can be seen as surrogate mi-
croorganisms, and are useful for the study of
specific opsonins and antigens. Various types
of coated beads have been used in phagocytosis
assays (reviewed in Bassøe et al., 2000): latex-
IgG, latex-IgG-C3, microspheres-C3b, micro-
spheres-iC3b, beads bearing ligands for
CD11b, latex-lysozyme, and OMV-beads.
Any of these coated beads might be used in
combined assays for monocyte and neutrophil
functions. OMV-beads mimic N. meningitidis.
Phagocytosis and oxidative burst mediated by
OMV-beads require the presence of specific
antimeningococcal antibodies and the appro-
priate antigen. REC and APS are used for com-
bined IgG-complement phagocytosis and IgG-
mediated phagocytosis, respectively. The same
principle can be applied to other antigens that
can be attached to polystyrene microspheres.
Attachment and internalization
Various agents have been used to discrimi-
nate attached from internalized particles. The
enzyme lysostaphin, which dissolves free and
attached staphylococci, does not penetrate neu-
trophils, and does not digest internalized bac-
teria. Crystal violet is used to quench attached
targets, but diffuses into phagocytes and
quenches intracellular targets too. For this rea-
son, crystal violet should be abandoned and
replaced with trypan blue, which does not enter
phagocytes.
Attachment
The percentage of phagocytosing PMNs and
MΦs is of the same order of magnitude before
and after trypan blue quenching. Thus, PMNs
and MΦs do not carry targets only on the
surface.
As a rule, 0.5 to 2.0 targets are attached to
the outer side of the plasma membrane.
Internalization
The remaining targets associated with
PMNs or MΦs are internalized. These results
are obtained whether phagocytosis is sustained
by PHS or complement is inactivated (PHS-
IN).
Current Protocols in Cytometry
Opsonins
There is ample proof that phagocytosis of
zymosan particles, many strains of S. aureus,
N. meningitidis, and many other bacteria spe-
cies does not occur in the absence of opsonins.
The importance of attaching opsonins to suit-
able antigens is underestimated. Passive ad-
sorption of opsonins to microspheres results in
feeble phagocytosis. Target/phagocyte ratios
>50:1 and incubation times >30 min are re-
quired to observe phagocytosis. Under these
conditions, percentage phagocytosis is only
≤15%, and the phagocytic index ∼1 to 3. This
low rate of phagocytosis is probably due to the
attachment of the Fcγ-part of the IgG molecules
to the microsphere surface, making them un-
available to Fcγ receptors on the leukocyte
surface.
Antigens
Opsonins bind to antigens attached to the
surface of targets. C3bi and the Fcγ-fragment
of IgG extend into the medium, making them
easily available for complement receptors and
Fcγ-R, respectively.
Phagocytic index
Flow cytometric measurements of the
phagocytic index have adapted the principles
introduced by Leishman (1927) and Maaløe
(1946). Leishman’s method is based on count-
ing the number of targets associated with each
cell. This is equivalent to measuring phagocyte
fluorescence along the fluorescence dimension
of the correspondingly labeled targets.
Maaløe’s method depends on counting the
number of free targets before and after phago-
cytosis. Leishman’s and Maaløe’s methods
have advantages and disadvantages.
(1) Leishman’s approach is applicable to
fluorescent microspheres. The stability of the
fluorescence is due to incorporation of dye in
the solid polymer. The fluorescence intensity is
unaffected by enzymes, toxic radicals, and pH.
Thus, the mean phagocytic index can be easily
and accurately calculated by dividing the mean
fluorescence of each phagocyte subpopulation
by the mean fluorescence of the free micro-
spheres. When only one subpopulation of
phagocytes is present, the phagocytic index
calculated from microsphere fluorescence
closely correlates with the index obtained from
the loss of particles from the suspension
(Lehmann et al., 1997).
(2) Maaløe’s approach overcomes the prob-
lem that the phagocytic index cannot be quan- Studies of Cell
tified by the FITC fluorescence of the phago- Function
9.19.17
Supplement 21
 cytes containing FITC-labeled targets (Bassøe
et al., 1983). Although that approach adds some
technical inconveniences (see Troubleshoot-
ing), it is mandatory for accurate measure-
ments.
Critical Parameters
Fluorescence measurements
Phagocyte FITC fluorescence has been used
as a measure of phagocytosis. This is a simple
approach that gives an overall impression of the
combined fluorescence of attached and inter-
nalized targets. The phagosomal environment
profoundly affects target fluorescence. Some
fluorescence changes persist after the targets
are liberated from the phagocytes (Bassøe,
1984; Hurst et al., 1984). In order to overcome
the influence of phagosomal pH on fluorescent
labels like FITC, phagocytes have been fixed
and permeabilized (Cantinieaux et al., 1989).
However, FITC molecules may be chemically
modified by the oxidative burst and other intra-
cellular chemical reactions (Hurst et al., 1984).
Fluorescent molecules attached to digestible
targets may be released from the targets by
phagosomal enzymes (Bassøe, 1984). Perme-
abilization may liberate digested, small fluo-
rescent amino acids, peptides, and glycosami-
noglycans from the cells. However, digestion
can barely be measured with <15-min incuba-
tion, and only a few fluorescent molecules are
expected to be released with shorter incubation
times.
Fluorescence measurements may underesti-
mate phagocytic indices. In general, the esti-
mation of the phagocytic index from the fluo-
rescence of digestible targets or pH-sensitive
fluorescent probes is discouraged.
QUICK-MIX controls
The fluorescence of FITC molecules is in-
fluenced by serum factors other than electro-
lytes. Proteins, for example, may diminish the
fluorescence intensity of FITC. The controls
overcoming these problems are nicknamed
QUICK-MIX. QUICK-MIX controls are not
subject to phagocytosis, but all the other incu-
bation steps are followed. There is only one
exception. QUICK-MIX is not required for
trypan blue–quenched samples of zymosan
particle phagocytosis incubated in HYPO-IN
since the leukocytes in these samples do not
phagocytose at all.
Assessment of
Phagocyte
Functions by
Flow Cytometry
9.19.18
Supplement 21
Phagosomal pH
Phagocyte FITC fluorescence depends on
quenching, digestion and release of florescent
molecules (amino acids, peptides, proteins, and
glucosaminoglycans) from stained targets,
electrolyte composition and concentrations,
and pH.
Clearly, the estimation of phagosomal pH
using FITC fluorescence should be interpreted
with care. However, little digestion of phago-
cytosed material takes place during the first 15
min of incubation (Bassøe, 1984). At 15 min,
the modal FITC fluorescences of liberated and
free FITC-labeled particles are similar (Bassøe,
1984). At 7.5 min, the fluorescence intensity of
liberated, FITC-stained targets is similar to that
of the free FITC-labeled particles. Thus, within
this short incubation time, the changes in FITC
fluorescence of phagocytosed targets are re-
versible. FITC fluorescence is a sensitive pH
indicator (Bassøe, 1984). Accordingly, with
incubation times <15 min, total phagocyte fluo-
rescence can be used as a (semiquantitative)
measure of phagosomal pH.
Mixing
The frequency of hits between targets and
phagocytes influences the rate of phagocytosis,
and the phagocytosis index is profoundly af-
fected by mixing (Bassøe, 2000). Several mix-
ing procedures have been used: periodical stir-
ring, end-over-end rotation, shaking, and auto-
mated microtiter plate mixers.
Periodical stirring and shaking are unde-
fined terms and should be avoided. End-over-
end rotation is used in flow cytometry phago-
cytosis assays to secure optimal mixing. The
disadvantage is the large volume and high cell
numbers (∼5 × 106 phagocytes) required. In
order to miniaturize phagocyte assays, and at
the same time standardize mixing, use 96-well
microwell plates and standard mixing.
Considering its importance, mixing of tar-
gets and leukocytes is taken on too easily. Vast
differences are reported in assays with corre-
sponding concentrations and activities of tar-
gets, phagocytes, and opsonins, but differences
in mixing (see Bassøe, 2000). Some commer-
cially available mixers are easy to use, the rate
of mixing can be controlled, and temperature
is the same in all microwells. Other mixers are
more laborious to use and temperature is not
reproducible from one well to the next. The two
following shakers work well and are recom-
mended:
Current Protocols in Cytometry
 (1) IKA-Minishaker MS 1 (IKA-Works) is
not temperature controlled, and was placed in
a incubator at 37°C.
(2) iEMS incubator/shaker (Thermo Lab-
systems) contains different shaking levels and
microtiter plates are temperature controlled.
Incubation time
The rate of phagocytosis is astonishingly
high. Non-lymphocytes (PMNs + MΦs)
phagocytose ∼80 S. aureus bacteria each within
15 min, and engulf ∼6 to 9 OMV-beads in 7.5
min. The phagocytic index increases with in-
creasing incubation time, but if the incubation
time is prolonged beyond the point when all
targets are phagocytosed, a diminished rate of
phagocytosis may be missed.
When microspheres are used, the phago-
cytic index can be accurately calculated from
the fluorescence values immediately from the
start of incubation. A short incubation time,
e.g., 5 min, might seem useful because at that
point the rate of phagocytosis is maximal.
Short incubation times pose problems for
Maaløe’s method. With short incubation times,
only a small fraction of the bacteria and zy-
mosan particles are phagocytosed, leaving only
a small difference between the initial and final
counts of free targets. Such a small difference
between target/phagocyte ratios is hard to
measure by Maaløe’s method, and is quantita-
tively unreliable. In the author’s hands, the
minimum incubation time with initial tar-
get/phagocyte ratio of 20:1 is 7.5 min. When
target/phagocyte ratio is 10:1, the differences
in the initial ratios and those following phago-
cytosis become prominent after 7.5 min.
Signal collection
A defined collection time minimizes the
drift in fluorochrome distribution and fluores-
cence intensity between samples. Collect data
for 30 sec. Signals are typically recorded from
1500 neutrophils, 100 to 200 monocytes, and
24,000 microspheres.
Sample sequence
In order to utterly reduce possible drift,
analyze the contents of two parallel microwell
series in opposite sequence.
Discrimination of PMNs from MΦs
The phagocytosis of microspheres increases
the SS of MΦs. Therefore, phagocytosing MΦs
cannot be discriminated from phagocytosing
PMNs by light scatter alone. Staining with
antiCD14-PE-Cy5 discriminates PMNs from
Current Protocols in Cytometry
MΦs also after phagocytosis. An exception is
cells from patients with paroxysmal nocturnal
hemoglobinuria (PNH), who lack CD14. For
these patients, phagocytosing MΦs cannot be
discriminated from PMNs by staining for
CD14.
Digestion
The measurement of digestion relies on
fluorescence of liberated targets. Therefore,
target concentrations and incubation times
must be chosen so that most of the targets are
internalized.
Two to three targets adhere to cell nuclei
after exposure to the detergent NP40 in the
Vindeløv’s solution. Because the number of
adherent particles is unknown, the fluorescence
of these targets cannot be used to measure
digestion.
Troubleshooting
Hemolysis
Lysis of red blood cells is mandatory since
red blood cells interfere with measurements by
blocking the leukocyte access to targets. If the
pelleted leukocyte suspension is red after the
first NH4Cl lysis, lysis should be repeated.
Cell clumping
Earlier methods relied on Hank’s buffered
salt solution supplemented with albumin, or
other buffers. Buffers containing Ca2+ and
Mg2+ promote leukocyte aggregation. Cell
clumping is overcome by washing leukocytes
in DPBS-GA. Note that Ca2+ and Mg2+ are
omitted from the solution. Cells can be kept in
DPBS-GA on ice for a few hours without af-
fecting phagocyte functions.
Cell clumping may occur during phagocy-
tosis. If so, the number of aggregates can be
measured using FS versus SS. The percentage
of cell clumps should be the same before and
after phagocytosis. Results should be discarded
if the fraction of doublets and clumps before
and after phagocytosis is not of the same order
of magnitude.
Adhesion to the sample flow lines in the
flow cytometer
Adhesion of targets and leukocytes to the
lines of the sample flow system is minimized
by diluting the suspensions in EDTA and by
keeping the samples on ice until the measure-
ment. Microspheres, E. coli, S. typhi, Str.
epidermidis, S. aureus, and zymosan particles Studies of Cell
do not adhere. However, some bacteria, e.g., Function
9.19.19
Supplement 21
 meningococci, adhere to sample flow lines.
This causes a loss of targets as they pass through
the fluidics system, and decreases the measured
target/phagocyte ratio. The phenomenon is ob-
served as a target/phagocyte ratio higher after
phagocytosis than before. Percentage phagocy-
tosis can still be measured accurately, but not
the phagocytic index. The problem can be less-
ened and possibly eliminated by diluting the
samples to avoid coincidence, and increasing
the sample flow rate. Controls using micro-
scopic counts are required.
Spontaneous phagocytosis
Some albumin batches are contaminated, in
particular, with endotoxin that can elicit phago-
cytosis and oxidative burst, and affect intracel-
lular processing. To avoid this problem, pur-
chase a small amount of albumin, record the
batch number, and test the albumin in the ab-
sence of serum to assess unwarranted phagocy-
tosis and oxidative burst. Compare phagocyte
FITC fluorescence after the phagocytosis of
FITC-labeled targets in the absence and pres-
ence of opsonins. If the batch works as ex-
pected, purchase and use this albumin batch.
Spontaneous oxidative burst
DHR in solution is slowly converted to
R123. If control neutrophils that have not been
phagocytosing microspheres stain green, indi-
cating R123 fluorescence, this staining is prob-
ably due to the conversion of DHR to R123
before phagocytosis. Make a fresh, new DHR
solution.
Trypan blue quenching
Several reports in the literature concern in-
ability to quench FITC fluorescence with try-
pan blue. Different batches of trypan blue—
even from the same company source—have
been reported to vary in quenching ability. This
problem is related not to trypan blue, but to the
relative concentration of trypan blue to the
intensity of target FITC staining. Therefore, the
staining procedure is standardized using a de-
fined optical density of the bacteria to be FITC
stained. The ability of trypan blue to quench
target FITC fluorescence is easily monitored
by flow cytometry.
If effective quenching of the targets cannot
be performed, targets should be stained less
intensively. The FITC staining protocol given
above allows effective quenching of FITC-la-
Assessment of beled bacteria and zymosan particles by trypan
Phagocyte
Functions by blue. Another problem with trypan blue is
Flow Cytometry clumps. Trypan blue clumps should be first
9.19.20
Supplement 21
removed by a crude filter followed by sterile
filtration (0.22-µm pore size).
Phagocytic index
This problem regards the differential count
of leukocytes and targets. When the tar-
get/phagocyte ratio is >20:1, the relative counts
of leukocytes and targets become inaccurate.
For example, with target%/leukocyte%, one
may have a 96%:4% ratio of 24, a 95%:5% ratio
of 19, and a 94%:6% ratio of 15.7. Thus, a small
measured difference in the percentage of targets
from 96% to 94% shifts the (apparent) target
load from 24:1 to 15.7:1. This change in target
load profoundly affects phagocyte fluores-
cence. Accuracy is increased by lowering the
ratio of targets to phagocytes. Therefore, the
determination of the concentration of targets
should be based not on a single measurement,
but repeated measurements of target/leukocyte
ratios using several target dilutions.
Using a low target/phagocyte ratio dimin-
ishes the strain on the phagocytes, and increases
the danger of statistical type 1 error. With small
targets such as bacteria and microspheres of
similar size, a target/phagocyte ratio of 20:1 is
useful for many purposes.
No phagocytosis
Proteins and other chemicals can adhere to
various tubes used for reagent storage. Use
polypropylene tubes that do not absorb essen-
tial ingredients such as opsonins.
Anticipated Results
Percentage phagocytosis
The measurement of the percentage of
phagocytosing leukocytes was introduced by
Hamburger in 1927, and has since been a pa-
rameter of choice (Bassøe et al., 1983). Quad-
rant statistics must be set with care to monitor
percentage phagocytosis. Usually, phagocytes
are at least five times more fluorescent than free
targets. Non-phagocytosing leukocytes are less
fluorescent than individual free targets. There-
fore, when the vertical quad-stat line is set just
above the fluorescence of the targets, phago-
cytes are clearly discriminated from non-
phagocytes. Since non-phagocytosing leuko-
cytes do not fluoresce at all, the discriminator
monitors an all-or-none phagocytosis response.
The all-or-none discriminator is useful for
the monitoring of receptor deficiencies and lack
of receptor function. About 96±3% of periph-
eral blood neutrophils phagocytose S. aureus,
zymosan particles, and OMV-beads and in
Current Protocols in Cytometry
HYP, PHS, and REC, respectively. Percentage
phagocytosis drops to ∼70% in REC-IN with
OMV-beads as target and is 0% to 5% in HYP-
IN and with zymosan particles as target. Per-
centage phagocytosis of S. aureus depends on
the PHS. It varies between 20% and 70% in
PHS-IN, but is stable for a long time for any
one PHS batch. This implies that complement
and IgG alone sustain phagocytosis by ∼96±3%
and 70% of the peripheral blood neutrophils.
Percentage phagocytosis by MΦs is ∼70%
to 75% with S. aureus, zymosan particles, and
OMV-beads in HYP, PHS, and REC, respec-
tively. The value drops to the order of 5%, 10%
to 50%, and 20% in HYP-IN, PHS-IN, and
APS, respectively.
These results are obtained in spite of the
regular observation that all PMNs from the
same suspensions carry FcγRIII (CD16) and
FcγRII (CD32), and all the MΦs carry CD32
and FcγRI (CD64). Under the same conditions
none of the PMNs carries CD64, and MΦs do
not express CD16. Thus, there is an expected
discrepancy between receptor expression and
the ability to phagocytose by the same recep-
tors.
Phagocyte subpopulations
One advantage of using fluorescent micro-
spheres is their low CV of ∼1.0, as compared
to the CV of FITC-labeled bacteria or zymosan
particles, which is ∼30% to 40%. The latter high
CV makes impossible the accurate discrimina-
tion of subpopulations of phagocytes differing
in fluorescence, whereas such a discrimination
between cell subpopulations is possible with
microspheres.
When leukocytes and OMV-beads are incu-
bated with inactive serum, the neutrophils do
not associate with OMV-beads. In REC, nearly
all neutrophils are phagocytosing. The percent-
age oxidative phagocytosis and the phagocytic
index vary within narrow limits. The difference
between percentage phagocytosis and percent-
age oxidative burst, i.e., percentage in region 4
minus percentage in region 1, is of the order
0.6%. The fraction of non-phagocytosing neu-
trophils is ∼1.5%. The fluorescence of the
OMV-beads is stable for several years.
In APS, monocytes are mainly localized to
region 1. In REC, the majority of monocytes
are observed in region 2. Very few MΦs are
resting, i.e., in region 3.
With APS and in the presence of DHR,
PMNs do not display green fluorescence.
Phagocytosis is triggered in REC, and a single
population is observed.
Current Protocols in Cytometry
In the absence of DHR, monocytes do not
fluoresce in the green. When incubated with
DHR 7.5 min at 37°C, monocytes become
green fluorescent even in the absence of serum,
OMV-beads, mixing, and antiCD14. The per-
centage of monocytes in region 1 is 89±9%.
AntiCD14 does not influence the monocyte
R123 fluorescence.
The oxidative ratio is a derived parameter
that informs on the conversion of DHR to R123
per ingested bead. MΦs start with a higher
R123 fluorescence than do neutrophils. With
increasing incubation time, the monocyte oxi-
dative ratio declines, whereas that of PMNs
increases. After 15 min, the values are of the
same order of magnitude in both cell types.
Chemotaxis
The controls contain lymphocytes, mono-
cytes, and neutrophils in proportion to the pe-
ripheral blood counts. Following spontaneous
migration and chemotaxis, only neutrophils are
observed in the FS versus SS cytograms. Ex-
pected spontaneous migration is ∼30% to 50%,
and chemotaxis 60% to 90%. Chemotaxis val-
ues depend on the concentration and activity of
ZAS and other chemotactic agents (IL-8, fMLP,
LTB4, and others).
Time Considerations
The whole procedure, i.e., Basic Protocols
1 to 4, is a demanding 1-day workload. Four
parallel samples can be investigated in 1 day,
but it is advisable to select those parts of the
individual protocols that are suited for the pur-
pose at hand. In Basic Protocol 1, e.g., a suitable
bacteria strain can be selected for study, perhaps
from a patient, and zymosan particles and mi-
crospheres can be ignored. Likewise, comple-
ment receptors and serum complement opsonic
activity can easily and rapidly be investigated
by using zymosan particles alone. Micro-
spheres are useful for the investigation of spe-
cific antigens, opsonins, and respiratory burst.
However, oxidative burst can also be studied
using unstained, non-fluorescent microorgan-
isms and zymosan particles.
After the addition of the sample to Vin-
deløv’s solution, the EB and FITC staining of
targets are stable at least 2 to 3 days in the dark
at 4°C. Flow cytometric investigations can
therefore be delayed.
Studies of Cell
Function
9.19.21
Supplement 21
 Literature Cited
Bassøe, C.-F., Laerum, O.D., Glette, J., Hopen, G.,
Haneberg, B., and Solberg, C.O. 1983. Simulta-
neous measurement of phagocytosis and
phagosomal pH by flow cytometry: Role of pol-
ymorphonuclear neutrophilic leukocyte gran-
ules in phagosome acidification. Cytometry
4:254-262.
Bassøe, C.-F. 1984. Processing of Staphylococcus
aureus and zymosan particles by human leuko-
cytes measured by flow cytometry. Cytometry
5:86-91.
Bassøe, C.-F. 2000. Flow cytometric quantitation of
phagocytosis in acute myelogenous leukemia.
Acta Haematol. 102:163-171.
Bassøe, C.-F., Smith, I., Sørnes, S., Halstensen, A.,
and Lehmann, A.K. 2000. Concurrent measure-
ment of antigen- and antibody-dependent oxida-
tive burst and phagocytosis in monocytes and
neutrophils. Meth. Enzymol. 21:203-220.
Assessment of
Phagocyte
Functions by
Flow Cytometry
9.19.22
Supplement 21
Cantinieaux, B., Hariga, C., Courtoy, P., Hupin, J.,
and Fondu, P. 1989. Staphylococcus aureus
phagocytosis. A new cytofluorometric method
using FITC and paraformaldehyde. J. Immunol.
Methods 121:203-208.
Hurst, J.K., Albrich, J.M., Green, T.R., Rosen, H.,
and Klebanoff, S. 1984. Myeloperoxidase-de-
pendent fluorescein chlorination by stimulated
neutrophils. J. Biol. Chem. 259:4812-4821.
Lehmann, A.K., Halstensen, A., Holst, J., and
Bassøe, C.-F. 1997. Functional assays for evalu-
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Immunol. Methods 200:55-68.
Contributed by Carl-Fredrik Bassøe
Haukeland University Hospital
Bergen, Norway
Current Protocols in Cytometry