DR ZEENAT

ASSITT PROFESSOR
PATHOLOGY
 Apoptosis
• A mode of rapid cell death after
irradiation
• “Programmed” cell death that is
potentially a controllable process
• Plays an important part in
embryogenesis and in tissue
regeneration
Relationship between apoptosis
and cell kill by radiation

Lymphomas 50%-60%
Solid tumors 10%-30%
Sarcomas < 2-3%

Apoptotic death starts 3-5 hours after irradiation

Cell loss factor in tumor growth kinetics

 Distinctive features of
Apoptosis
• Chromatin condensation
• Degradation of DNA-ladder form
• Cell shrinkage
• Scattered throughout a tissue
• Inflammation is absent
• Activation of a specific program
of genes
 Necrosis Apoptosis

• Accidental death • Programmed death

• Severe & sudden injury • Process is more subtle, and

ischaemia, physical or more physiologically
chemical trauma determined.
• Cellular and organelle
• Cell shrinkage
swelling
 Necrosis Apoptosis

• Random spillage of • Plasma & nuclear membrane

cellular content blebbing
organelle relocalization &
• Inflammatory response chromatin condensation

• Production of membrane
• Major site of damage
enclosed “apoptotic bodies”
plasma membrane

• Clear by macrophages

• No inflammatory response
NECROSIS

A B C

APOPTOSIS
Thio proteases
Detection of Apoptosis by Flow
Cytometry

• Early stage Annexin V/7-AAD(PI)

• Mid stage TUNEL assay

• Late stage < Go/G1 DNA content

 B
C
A

Apoptosis D

F
E
Annexin V: An Early Marker of Apoptosis

One of the earliest indications of apoptosis is

the translocation of the membrane phospholipid
phosphatidylserine (PS) from the inner to the
outer leaflet of the plasma membrane.

Once exposed to the extracellular environment,

binding sites on PS become available for
Annexin V, a 35-36 kDa, Ca 2+-dependent,
phospholipid binding protein with a high affinity
for PS.
Annexin V

The translocation of PS precedes other

apoptotic processes such as loss of
plasma membrane integrity, DNA
fragmentation, and chromatin
condensation.
As such, Annexin V can be conjugated to
biotin or to a fluorochrome such as FITC,
PE, APC, Cy5, or Cy5.5, and used for the
easy, flow cytometric identification of cells
in the early stages of apoptosis.
Annexin V

Because PS translocation also occurs

during necrosis, Annexin V is not an
absolute marker of apoptosis.

Therefore, it is often used in conjunction

with vital dyes such as 7-amino-
actinomysin (7-AAD) or propidium iodide
(PI), which bind to nucleic acids, but can
only penetrate the plasma membrane
when membrane integrity is breached, as
occurs in the later stages of apoptosis or
in necrosis.
Annexin V
No Apoptosis = Cell Viability

Cells that are negative for both Annexin V and the

vital dye have no indications of apoptosis: PS
translocation has not occurred and the plasma
membrane is still intact.

Early Apoptosis

Cells that are Annexin V-positive and vital dye-

negative, however, are in early apoptosis as PS
translocation has occurred, yet the plasma
membrane is still intact.
Annexin V

Late Apoptosis or Cell Death

Cells that are positive for both Annexin V and the

vital dye are either in the late stages of apoptosis or
are already dead, as PS translocation has occurred
and the loss of plasma membrane integrity is
observed.

When measured over time, Annexin V and a vital dye

can be used to monitor the progression of
apoptosis: from cell viability, to early-stage
apoptosis, and finally to late-stage apoptosis and
cell death.
 4.72 56.69

93.82 41.84

1.46 2.37

12.67 61.22

37.02
85.25

2.08 1.76
Apoptosis

TUNEL ASSAY
TdT-mediated dUTP Nick-End Labeling
DNA degradation

Incorporation of fluorescein-12-dUTP to
3’-OH DNA ends using
enzyme Terminal deoxynucleotidyl Transferase
(TdT)
****
dUTP
5’ 3’
OH
Apoptosis
TUNEL Assay
with BrdUrd labeling
anti-BrdUrd antibody

• Cell number (2 x 106)

• Chromatin denaturation
– Acid
– Pepsin
– Separation between neg. and pos. signals
• Data analysis
• TUNEL assay
 10 min 30 min
103

ANTI-BrdUrd ANTIBODY (FITC) S

102

101

100
G2M

G1
-1
10
0 10 20 30 40 50 60 0 10 20 30 40 50 60

DNA CONTENT (PI)

In vitro: 10 mM BrdUrd
In vivo: 10 mg/Kg BrdUrd IP
 HL60 cells

control 2.0 mg/ml camptothecin

Apoptotic cells
TUNEL

DNA CONTENT
 Apoptosis (TUNEL) from Rat Lavage Fluid

Control, 11 months Sterling V, 11 months

Anti-BUDR Antibody

Anti-BUDR Antibody
0.97 % 20.82%
(TUNEL)

(TUNEL)
G1
G1
Cell number

Cell number
Sub-G1

S
G2M S
G2M

DNA Content DNA Content

 Apoptosis
Less than Go/G1 DNA content

• Gatings
– FS : intact cells
– DNA (area) vs. DNA (width)
– DNA histogram
 R2
R1

M1

R3

Histogram Statistics

File: NCI 520 0h.010 Log Data Units: Linear Values

Sample ID: NCI 520 TA 0h Acquisition Date: 31-May-0
Gate: G3 Gated Events: 8751
Total Events: 10000 X Parameter: FL3-A (Linear)

MarkerLeft, RightEvents % Gated% Total Mean

All 0, 1023 8751 100.00 87.51 322.23
M1 0, 226 21 0.24 0.21 183.86
 R2
R1

M1

R3

Histogram Statistics

File: NCI 520 TA 50nM 12h.004 Log Data Units: Linear Values
Sample ID: NCI 520 TA 50nM 12h Acquisition Date: 31-May-0
Gate: G3 Gated Events: 6557
Total Events: 10000 X Parameter: FL3-A (Linear)

MarkerLeft, RightEvents % Gated% Total Mean

All 0, 1023 6557 100.00 65.57 349.55
M1 0, 226 1407 21.46 14.07 151.67
 R2
R1

M1

R3

Histogram Statistics

File: P21+/+12Gy Log Data Units: Linear Values

Sample ID: Acquisition Date: 05-Aug-02
Gate: G3 Gated Events: 10907
Total Events: 16141 X Parameter: FL3-A (Linear)

MarkerLeft, RightEvents % Gated% Total Mean

All 0, 1023 10907 100.00 67.57 338.40
M1 5, 154 289 2.65 1.79 102.71
 Equipment

• Tissue culture equipment

– Sterile tissue culture hood, Incubator (CO2,
37oC, humidified), Microscopes( upright,
inverted, dissecting), Pipettes,
Hemacytometers (Coulter counter).
• Flow cytometer
• Radiation source
 Supplies
• 75% cold (4oC) ethanol
• 1x phosphate buffered saline (PBS)
• RNase solution in PBS (1 mg/ml)
• Propidium iodide solution (20 g/ml)
• APO-BRDU kit, AU1001, Phoenix Flow
• Annexin V/PI kit, PF032, Calbiochem
• 15 ml conical centrifuge tubes
• Pipettes (1ml, 5 ml)
• Sample tubes (Falcon 35 2058, 35 2235-filter)
• 100 MB Mac formatted zip-disks
Procedures: sample preparation
for PI staining(<Go/G1)

• Cell permeablization
– fresh cells: hypotonic buffers, detergents
– fixed cells: 75% EtOH overnight
1x106 cells in 3 ml EtOH
• RNase: 1 mg/ml 30 min at room temp.
• DNA/PI ratio: 20 g/ml for 1x106 cells
• Cell concentration (1x106/ml)
Procedures: 10 Gy

TUNEL assay

TdT + BrdU

Anti-BrdU antibody
 Procedure: Annexin V/PI(7AAD)
• Resuspend at least 0.5 x 106 cells in 12x75mm
tubes at a concentration of 106 cells/ml.
• Add 2ml of cold PBS to tubes.
• Centrifuge for 7 minutes at 1,000rpm.
• Resuspend pellet in 2ml of cold PBS.
• Centrifuge for 7 minutes at 1,000rpm.
• Resuspend cells in 0.1ml of 1x binding buffer.
• Add 10µl of FITC-conjugated annexin V and 5µl
of 7-AAD to tubes.
• Gently vortex.
• Incubate at room temperature for 15 minutes in
the dark.
• Add 0.9ml of 1x binding buffer.
• Analyze samples within 1 hour of staining.
Lab Demonstrations
room 3-4151
• Sample measurement
• Data analysis

• Treatment condition:
– 10 Gy, 15 Gy
– 0, 6, 24, 48 hr post-irradiation