Multicolor Panel Designing

for Flow Cytometry

David Mzinza, PhD | dmzinza@must.ac.mw
Important considerations
• Flow cytometry is a powerful tool for identifying and analyzing multiple antigens
simultaneously, however, increasing the number of antigens and fluorophores also
increases the complexity of the experimental design.
• It is very important to have a properly designed flow cytometry panel for accurate
identification of cell populations including rare cells.
• When designing a flow cytometer panel, four key considerations must be made
i. Biological question
ii. Research your fluorochromes
iii. Know the instrument you are using
iv. Optimise your panels
1. Biological question

Establishing a biological hypothesis

Having a clear question and hypothesis is crucial for choosing Panel
which markers to target to answer the biological question
Live dead
Question CD45
What is the impact of ART initiation in PLHIV on ART on T CD3
cell phenotype CD4
CD8
Hypothesis CD57
The initiation of ART in PLHIV leads to reduced CD4 and CD279 (PD-1)
CD8 T cell exhaustion and senescence and restoration of IFN-γ
function TNF-α
Cell type and antigen characteristics
Leucocyte antigens can be categorized based on their patterns of
expression

Primary CD4
Well-characterized; easily classified as positive or negative; typically defines broad
subsets or lineages
• Examples: CD3, CD4, CD19
Secondary CD45RA
Well-characterized; typically expressed at a higher density; often over a continuum
• Examples: CD27, CD28, CD45RA, CD45RO

Tertiary CD25
Expressed at low levels; variable upon activation; unknown, critical
• Examples: CD25, STAT5, FoxP3
2. Research your fluorochromes

Evolution of fluorochromes

• There are over 367 CD markers

• Intracellular proteins
• Cytokines
• Cell signalling
• Transcription factors
• Phosphoproteins
• The availability of fluorochromes
has driven major advances in flow
cytometry
Fluorochromes with corresponding lasers

Laser
1 BUV395
2 BUV496
3 BUV563
BUV496 BUV615-P BUV737 4 UV BUV615-P
UV BUV395 BUV563 BUV661 BUV805
5
6
BUV661
BUV737
7 BUV805
8 BV421
BV480
9
BV510
BV421 BV570 BV650 BV750 10 BV570
Violet BV480 BV605 BV711 BV786
11
12
Violet BV605
BV650
13 BV711
14 BV750
15 BV786
BB515
BB630-P BB700-P 16 FITC
AF488
Blue BB515 BB660-P BB790
17 BB630-P
18 Blue BB660-P
BB700-P
19 PerCP
PerCP-Cy5.5
20 BB790-P
PE-CF594 PE- BYG584-P
21
Green Cy5.5**
22
Yel-Grn
PE
PE-CF594
BYG584 PE-Cy5 PE-Cy7 23 PE-Cy5
24 PE-Cy5.5
25 PE-Cy7
APC
26
AF647
APC-R700 APC-R700
27 Red
Red AF700
APC-Cy7
APC APC-H7 28
APC-H7
 Fluorochromes Reveal Biology

PE PE-CF594 BV421 Alexa Fluor 647 • The choice of fluorochrome

helps to understand more
about the biology of the
experiment.
• Bright dyes are important
CD3

PerCP-Cy5.5 Alexa Fluor 700 V450 FITC when looking at dim antigens.

CD197 (CCR7)
• Many factors can influence the relative fluorochrome/reagent performance on a given
instrument, including laser power, PMT voltage, optical filters, antibody clone,
biological sample, and staining methodology.
3. Know your instrument

• Before designing your multicolour flow panel, Common lasers used in flow
you will need to determine the following cytometry
factors:
i. The number and type of lasers
ii. The number of detectors
iii. The type of filters available on your flow
cytometer
• You can only be able to use fluorophores
excited by the corresponding wavelength of
light from the laser
4. Panel optimisation

Resolution vs background
• Resolution is the degree to which a flow cytometer can “Negative” Dim Bright
distinguish dimly stained cells from unstained cells.
• There are many factors that can affect the resolution of dim
populations, but they can be broken down into various
sources
i. Negative population has low background;
populations well resolved
ii. Negative population has high background;
populations not resolved
iii. The negative population has low background but
high rSD (spread); populations not resolved
• The ability to resolve populations is a function of both
background and spread of the negative population.
Factors affecting resolution
Optical and Instrumentation Factors:
• Laser Power and Wavelength: The laser's power and wavelength used for excitation
can affect resolution by influencing the intensity and quality of the emitted
fluorescence.
• Optical Filters: The selection and optimization of optical filters can impact the
separation and detection of specific fluorochromes or markers.
Fluorochrome Selection and Fluorophore Characteristics:
• Fluorochrome Brightness: The brightness of the fluorochrome influences the intensity
of the emitted fluorescence and, thus, the resolution of positively stained
populations.
• Spectral Overlap: Spectral overlap of fluorochromes can reduce resolution, making it
difficult to distinguish distinct populations when multiple markers are used.
Sample Preparation and Staining:
• Staining Protocol: Properly optimized staining protocols, including incubation times
and concentrations of antibodies, are crucial for achieving optimal resolution.
• Cell Viability and Aggregation: Cell viability and the presence of cell aggregates can
affect the accuracy and resolution of the flow cytometry results.
Sample Characteristics:
• Cell Density: High cell density can lead to coincidence events, where more than one
cell passes through the laser at the same time, affecting resolution and accuracy.
• Cell Size and Complexity: The size and complexity of cells can impact how they
scatter light and affect resolution based on these parameters.
Stain Index: The Standard Metric of Resolution

•The staining index (SI) is used to measure spread

Brightness •The brightness is a function of the

• Reagent
Width of negative •Antigen density, fluorochrome used.
• Instrument
• PMT gain setting, laser power
•The width of the negative is a function of
– Instrument performance
BrightnessMFI – Spillover (multi-color panels)
Stain Index =
Width of NegativeSD
• SI is a quantitative measure used to assess the strength and specificity of staining for a
particular marker on cells.
• It helps in evaluating the quality of the staining and determining the separation between
positive and negative populations.
• SI is calculated using the mean fluorescence intensity (MFI) of the positive population
(usually the cells stained with the specific marker) and the MFI of the negative population
(cells without the specific marker), as well as the standard deviations of these populations.
(MFI of positive population)−(MFI of negative population)
• The formula for SI is:
2 × (SD of positive population + SD of negative population)

• A higher SI indicates a better separation between the positive and negative populations,
suggesting a more reliable and specific staining for the marker of interest.
Fluorescence spillover
• Fluorescence spillover is the single most important factor affecting resolution
sensitivity in multicolour flow cytometry experiments
• Fluorescence spillover from other channels:
• directly and irreversibly reduces the resolution sensitivity of that channel
• contributes to background
• This “background” is subtracted in the process called compensation.
 Spillover sources

FITC / PE
Adjacent Detectors

Residual Base Fluorescence BV786 / BV421

BUV737 / BV711
Similar Emission Spectra (Cross-laser)
How to deal with compensation issues?
FITC Stained Control
PE

FITC

Run single-stained compensation controls

• Important: if you have an experiment with 11 fluorochromes, it is
essential to prepare compensation controls for each fluorochrome used
Important considerations for compensation controls
4 key considerations for compensation:
1. Control and sample should use the same fluorophore (do not use A488
for FITC)
2. Compensation controls ought to be at least as bright or brighter than the
fluorochromes used in the sample
3. Background fluorescence for Positive and Negative controls should be
the same
4. Compensation controls MUST match the exact fluorochromes used in
the sample
Fluorescence Spillover Introduces Background / Spread Into Other
Detectors

• Fluorochromes spillover into other detectors;

e.g. PerCP-Cy™ 5.5 spills into the PE-Cy7
detector.
• This fluorescence spillover contributes to
• increased background (MFI)
• spread (rSD)

• A sample is correctly compensated when, in the spillover detector

(PE-Cy7), the MFI of the positive population is equivalent to that
of the negative population.
PE-Cy7

rSD

• However, the spread (rSD) introduced by the spillover is not

rSD removed by the compensation and reduces the resolution (SI) of
any double-positive cells.
PerCP-Cy5.5
Recognising over-compensated and under-compensated data

• Automatic flow cytometry compensation pipelines sometimes over-

compensates or under-compensates the data
• Under-compensation occurs when fluorescent signal from one detector has been
under-subtracted, leading to fluorescent signal leaning towards the centre of the
plot
• Over-compensation happens when fluorescence signal from one detector has
been over-subtracted, leading to fluorescent signal leaning towards the X-axis
 Over-compensation Under-compensation Properly compensated

In a properly compensated NxN plot, the median fluorescence of the positive

population should be equal to the median fluorescence of the negative population