Apoptosis assays detection and methods

Apoptosis Assays - Detection and
Methods
By,
Prabhu Thirusangu,
Research Scholar,
Sahyadri Science College,
Kuvempu University,
Shimoga
9th July,2013

Cell Viability and Death
• Functional assay
• DNA labeling assay
• Morphological assay
• Reproductive assay
• Membrane integrity assay

The major criteria employed in viability assay
Category of
viability assay
Assays Principles
Membrane integrity
assay
-Exclusion dyes
-Fluorescent dyes
-LDH leakage
-Annexin v assay
The determination of membrane integrity via
dye exclusion from live cells
Functional assay
-MTT, XTT assay
-Crystal violet/ Acid
phosphatase(AP) assay
-Alamar Blue oxidation-
reduction assay
- Neutral red assay
-[3H]-thymidin/ BrdU
incorporation
Examining metabolic components that are
necessary for cell growth
DNA labeling assay -Fluorescent conjugates
cell selection and viability assay
Morphological
mechanism based
assay
-Microscopic observation
-Caspase 3 detection
-PARP cleavage assay
Determination of morphological change
Reproductive assay -Colony formation assay Determination of growth rate

Principle
Trypan Blue dye Exclusion Methods

 Features distinguishing live from dead cells include the loss
of transport function across plasma membrane which results
from loss of membrane integrity.
 Cells must be counted within 3-5 min because the number of
blue-staining cells increases with time after addition of the
dye.
 Large numbers of samples have to be counted, it may be
inconvenient to perform all the tests on the same day by
counting one cell suspension at a time before staining the next
sample.
• - Viable cells : small, round and refractive
• - Non-viable cells : swollen, larger, dark blue
Exclusion dyes

 Ethidium bromide (EtBr) and propidium iodide (PI)
 PI binds to nucleic acids upon membrane damage :
flow cytometric techniques depend on fluorescence, PI
is ideally suitable for the rapid evaluation of the
permeability properties of large numbers of cells while
maintaining good statistical accuracy.
PI is impermeable to intact plasma membrane.
Intercalates with DNA or RNA  red
Fluorescent dyes

 Fluorescein diacetate (FDA) is a nonpolar ester which passes
through plasma membranes and is hydrolyzed by intracellular
esterases to produce free fluorescein, the polar fluorescein is
confined within cells which have an intact plasma membrane and
can be observed under appropriate excitation conditions.
 Undamaged cell : highly fluorescent fluorescein dye
 Damaged cell : fluoresce only weakly
 greenish-yellow at 450-480 nm

Intact cell –
PI and FDA is added
Fluorescein in
intact cells
Schematic illustration of the principle of
PI/FDA cell viability assay
● FDA (Fluorescein diacetate)
● PI (Propidium iodide)
Plasma membrane is damaged
; fluorescein leaks out
PI enters and strains
nucleic acids

• 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.
• 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

• 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
• Result
• annexin-/PI-, annexin +/PI-, annexin+/PI+ and annexin –/PI+

• 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.
• 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.

Apoptosis assays detection and methods

LDH ASSAY
• LDH catalyzes the reduction of NAD+ to NADH and H+ by oxidation of
lactate to pyruvate. In the second step of the reaction, diaphorase uses the
newly-formed NADH and H+ to catalyze the reduction of a tetrazolium salt
(INT) to highly-colored formazan which absorbs strongly at 490-520 nm.
Test principle
The assay is based on consideration that tumor cells possess high concentration of
intracellular LDH and the cleavage of a tetrazolium salt when LDH is present in the
culture supernatant.

 Quantitative value for the loss of cell viability
 The activity of LDH can be measured as the reduction of
pyruvate to lactate.
 The reduction is coupled to the oxidation of NADH to NAD+,
which is followed spectrophotometrically at 340nm
LDH
Pyruvate + NADH + H+ ⇌ NAD+ + lactate
 As NADH has a high absorbance at 340nm compared to NAD+,
the reaction is measured as the rate of decrease in absorbance at
340nm.
LDH (lactate dehydrogenase) Leakage

MTT Assay
Introduction
 This assay is a sensitive, quantitative and reliable
colorimetric assay that measures viability, proliferation
and activation of cells.
 The assay is based on the capacity of mitochondrial
dehydrogenase enzymes in living cells to convert the
yellow water-soluble substrate 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a
dark blue formazan product which is insoluble in
water.

 The amount of formazan produced is directly
proportional to number of viable cells present in the
sample.
metabolically active Cell
MTT Formazan
Insoluble

Materials and equipment
MTT solution (5 ㎎/㎖ in phosphate buffered saline (PBS) pH 7.5),
HCl, Propan-2-ol
96-well microtiter plate, ELISA reader
Procedure (suspension and monolayer cells)
1. Prepare MTT stock solution and filter through a 0.2 ㎛ filter to
sterilize and remove the small amount of insoluble residue
2. To 100 ㎕ cell suspension or cell monolayer in each microtiter well
add 10 ㎕ MTT
3. Incubate in a humidified incubator at 37℃ for 3 h
4. Add 100 ㎕ 0.04 M HCl in propan-2-ol to each well and mix
thoroughly to dissolve insoluble dark blue formazan crystals
5. Read plate on a ELISA reader using a test wavelength of 570 nm
and reference wavelength of 630 nm

Compare with MTT assay and XTT assay
Culture cells in a MTP
for a certain period of time (37℃)
MTT assay XTT assay
Prepare labeling mixture
Incubate cells (0.5-4 h, 37℃)
Add solubilizing solution
(Isopropanol) and incubate
Measure absorbance using an ELISA reader
Add XTT labeling mixtureAdd MTT labeling reagent
Insoluble formazan Soluble formazan

Example: MTT and XTT
MTT XTT
Jenny G., Mark H., Anna J., Inger K., Douglas Mc., Roland M., 2002.
Evaluation of redox indicators and the use of digital scanners and spectrophotormeter for
quantification of microbial growth in microplates. J. Micro. Methods. 50:63-73

Principle
 Dye elution:
Cell up-taken dye was measured colorimetric method after acetic
acid dye elution.
 Nuclei counting
Incubation of cell samples in a mixture of citric acid and crystal
violet causes cells to lyse and the released nuclei to stain purple.
Crystal violet

Procedure
 Dye elution
① After removal of medium, rinse
96 well plates with 100 ㎕/well
of PBS and stain with 100 ㎕
0.25% (g/10ml) aqueous crystal
violet for 10 min.
② Rinse plates four times in tap
water.
③ Dry the outsides of the plates
with paper to help avoid water
stains, and then dry the plates at
37℃. When dry, add 100 ㎕ per
well of 33% glacial acetic acid
(33 ml/100ml) and mix the
contents of each well before
reading at 570 nm.
 Nuclei counting
① Collect the cells from animal
and centrifuge it.
② Remove clear supernatant by
aspiration.
③ Add 1ml of crystal violet
reagent.
④ Incubate at 37℃ at least 1 h.
⑤ Introduce a sample into the
hemocytometer chamber and
count the purple-stained nuclei
as for whole cells.

Acid phosphatase (AP) assay
 The action of this enzyme in many of tissue is to cleave a
waste product called pyrophosphate and effectively
convert it to a useable phosphate.
 P-nitrophenyl phosphate will be the substrate and
nitrophenol is the product of this reaction.
 Nitrophenol is colorless but when the pH of the reaction
solution is alkaline, it is appears yellow. The pH of the
reaction solution will be changed by the addition of NaOH.

P-nitrophenyl phosphate + Acid phosphatase
 Nitrophenol + HPO4
-2

Materials and equipment
Substrate-containing buffer : 10 mM P-nitrophenyl phosphate in
0.1 M sodium acetate pH 5.5, 1 M NaOH
96-well micro titer plate, Microplate reader
Procedure
1. At end of cell growth period, remove medium and rinse wells in
100 ㎕ PBS
2. Add 100 ㎕ substrate-containing buffer to each well
3. Incubate for 2 h in incubator. Read plates at 405 nm, and either
reincubate for a further time if increased sensitivity is required, or
‘stop’ with addition of 50 ㎕/well of 1 M NaOH to cause an
electrophilic shift in the p-nitrophenol chromophore and thus
develop the yellow color, giving greatly increased sensitivity

 Principle
In the presence of cellular metabolism the color of Alamar Blue (ALB) changes
from a fully oxidized, non fluorescent blue to a fully reduced, fluorescent red.
ALB will be reduced by a variety of enzymes and small molecules, including
the cytochrome system, FMN, FAD, NAD, and NADP.
Advantages
Simple, rapid, inexpensive, required no lysis, extraction or washing of sample
 Disadvantages
Unstable during storage
 Characteristics
- Sensitivity :
- The ALB assay is faster, simpler, and less artifact prone than the MTT assay.
Alamar Blue oxidation-reduction assay

Procedure
① At the end of an experimental incubation period, add 1 vol of ALB stock solution
per 25 vols (4%v/v) of growth medium in each well (8 ㎕ ALB for 200 ㎕ of
growth medium)
② Incubate plates at 37℃ for 3 h to allow metabolic dye reduction.
③ Equilibrate plates to room temperature for 30 min in the dark.
④ Measure the relative fluorescence at 530~560 nm excitation and 590 nm emission
wavelengths. Fluorescence is temperature sensitive; either equilibrate plates in a
warm room at the culture incubation temperature.. The ratio of test to control
fluorescence values at 590nm measures the effect of a treatment on cell growth or
metabolism.
⑤ For spectrophotometric assays, correct for the spectral overlap of the oxidized
and reduced forms of ALB by measuring each sample at two different
wavelengths, between, approximately, 540~630 nm . One of these must be a low
wavelength (LW) and the other a high wavelength (HW); for example, 570 ~ 600
nm, respectively.

⑥ A correction factor (RO) for the absorbance of oxidized ALB must be
calculated.
ⓐ Measure the absorbance (AM) of growth medium alone. (no ALB)
ⓑ Measure the absorbances of oxidized (blue) ALB in growth medium at the low and
high wavelengths.
ⓒ Substract AM from each of the measured ALB absorbance to produce, respectively,
AOLW and AOHW , the absorbance of oxidized (blue) ALB at the low and high
wavelengths.
ⓓ Calculate the correction factor RO of oxidized ALB:
RO=AOLW/AOHW
⑦ Measure the absorbance values (ALW and AHW) of a test sample at each
wavelength.
⑧ Calculate the percentage of reduced ALB (ARLW) in a sample as:
ARLW = 100 x [ALW-(AHW x RO)].
⑨ Calculate the percentage difference in reduction (PDR) between treated
and control cells: PDR = 100 x (test ARLW/ARLW for positive growth control)

Neutral Red assay
(3-amino-7dimethyl-2-methyphenazine hydrochloride)
 Principle
- The incorporaton of NR into the lysosomes of viable
cells after their incubation with test agents.
 Use
- Industrial, pharmaceutical, environmental and other
testing laboratories concerned with acute toxicity
testing.
 Advantages
- Simplicity, speed, economy, and sensitivity

Materials and Equipments
 Solution
① Neutral red
4mg/ml stock solution
Dilute 1:100 into medium , incubate
overnight at 37℃ and centrifuge for
10 min at 1500 g before use.
② 1% CaCl2/0.5% formaldehyde
Mix 6.5 ml 37% formaldehyde with
50 ml 10% CaCl2 and 445 ml distilled
water.
③ 1% acetic aicd/50% ethanol
Mix 4.75 ml acetic acid with 250 ml
95% ethanol and 245 ml distilled
water.
 Equipment
① Complete media suitable for
chosen cell type.
② Culture petri dish
③ 96well tissue culture plate
④ Inverted microscope
⑤ ELISA-type
spectrophotometer
⑥ Microplate shaker
⑦ Eight-channel pipette

Procedure
① Resuspend cells of actively growing culture and count cells and accurately
allocate appropriate number suspended in medium.
② Seed 0.2 ml containing desired number of cells to each well of 96 well plate and
incubate at 37℃ for 24 h or longer.
③ Remove the medium and add fresh medium containing graded dilutions of test
agent. Incubate for desired length of time. Examine at least 4-8 wells per
concentration of test agent.
Keep serum concentration as low as possible during this step.
④ After incubation for desired time interval, remove medium with test agent and
incubate cells with fresh medium containing 40 ㎍/ml NR dye.
⑤ Continue incubation for 3h to allow for incorporation of vital dye into survival
cells.
⑥ Remove medium by inverting the plate and rapid rinse with a mixture of 1%
CaCl2 / 0.5%formaldehyde.
⑦ Extract dye into supernatant with 0.2 ml of solution of 1% acetic acid/50%
ethanol.
After10 min at room temperature and rapid agitation for a few seconds on a
micrometer plate shaker, scan the plate with an ELISA-type spectrophotometer
equipped 540 nm filter.

DNA labeling assay
(using fluorescent probes assay)

Principle
The rate of DNA synthesis is a reflection of
proliferation under many condition.To measure the
proliferative rates by [3H]-thymidine uptake, cells
are cultured in microtitre wells, thymidine is
added, and the uptake by DNA is measured , after
lysing and washing on, by scintillation counting.
Bromodeoxyuridine(BrdU) can be incorporated
instead of [3H]-thymidine and the incorporation
can be assayed with antibodies to BrdU in a non-
radioactive assay.
[3H]-thymidine and BrdU incorporation
(DNA synthesis measurement)

Schematic diagram of [3H]-TdR and BrdU

Labeling index with [3H]-thymidine
① Set up the culture at 2x104 cells/ml~ 5x104 cells/ml in 24 well plates
containing cover-slips. Grow to the desired cell density.
② Add [3H]-thymidine to the medium and incubate the cultures for 30
min.
③ Remove the labeled medium, and discard it into a designed container
for radioactive waste.
④ Wash the cover-slips three times with PBSA.
⑤ Add 1:1 PBSA: acetic methanol, 1ml per well, and remove it
immediately
⑥ Add 1ml of acetic methanol to each well, and leave the cultures for
10min.
⑦ Remove the cover-slips, and dry them with a fan
⑧ Mount the cover-slip on a microscope slide with the cells uppermost.
⑨ Leave the mountant to dry overnight.

37
Terminal Deoxyribonucleotidyl Transferase-
Mediated dUTP Nick End Labeling (TUNEL) assay
* Terminal deoxyribonucleotidyl transferase (TdT) can
catalyze the addition of nucleotide at 3’ OH end of DNA.
* Cells are incubated with TdT, Co2+,
biotinylatedmdeoxyuridine triphosphate.
* Cells are then incubated with fluorescently labeled
streptavidin.
*Apoptotic cells can be detected by fluorescence microscopy
or flowcytometry.

Incorporation of fluorescein-dUTP to
3’-OH DNA ends using
enzyme Terminal deoxynucleotidyl Transferase (TdT)
5’ 3’OH
dUTP****
TUNEL ASSAY
TdT-mediated dUTP Nick-End Labeling
Detection of DNA Degradation

dNTP dUTP
Direct Indirect
X Fluoresein, FITC, PE etc Biotin DIG
Avidin conjugated with
fluoresein, AP, POD
Anti-DIG antibody
conjugated with fluoresein,
AP, POD
Ⅰ. Enzymatic DNA labeling

Comet Assay
The single cell gel electrophoresis (SCGE), the
Comet Assay, is a fairly simple procedure by using a
micro gel and electrophoresis to detect DNA damage.
After image analysis the damage in DNA looks like a
comet, so that’s why it is known as Comet Assay. This
technique is further developed and introduced the use of
high alkaline conditions. This step increased the ability
of the assay to detect not only the double strand breaks
but also the single strand breaks.
To detect genotoxicity of the drug

Treatment
Single cell
suspension
Slide
preparation
Alkaline
lysis
DNA
unwinding
Image
analysis
Alkaline
electrophoresis
Stain
DNA-Repair
Flow chart for both
methods

• Tail length:
It is defined as a measurement from the point of
greatest intensity within the comet head.
• Tail moment:
It is defined as the product of the tail length and
the fraction of total DNA present within the tail.
TM = tail length*100
head
• Tail intensity:
It is defined as the florescence detected by image
analysis in the tail, which is proportional to the amount
of DNA that has moved from the head region into the
comet tail.

PARP CLEAVAGE ASSAY: WESTERN BLOT AND FLOW CYTOMETRY
Caspase-3 cleaves many cellular proteins including PARP (Poly (ADPRibose)
Polymerase). PARP is a 116 kDa nuclear protein which is strongly activated
by DNA strand breaks
.
PARP plays a role in DNA repair as well as in other cellular processes,
ncluding DNA replication, cell proliferation and differentiation.
During apoptosis,caspase-3 and -7, cleave PARP to yield an 85 kDa and a 25
kDa fragment. PARP cleavage is considered to be one of the classical
characteristics of apoptosis.
An anti-PARP-FITC conjugated Cleavage Site-Specific Antibody (CSSA) (Cat.
#44-699) that can detect apoptotic cells by flowcytometry. An alternative to
the TUNEL assay, the PARP-FITC CSSA can detect apoptosis in adherent and
suspension cells.

PARP CLEAVAGE ASSAY: WESTERN BLOT AND FLOW CYTOMETRY
Caspase-3 cleaves many cellular proteins including PARP (Poly (ADPRibose)
Polymerase). PARP is a 116 kDa nuclear protein which is strongly activated
by DNA strand breaks
.
PARP plays a role in DNA repair as well as in other cellular processes,
including DNA replication, cell proliferation and differentiation.
During apoptosis,caspase-3 and -7, cleave PARP to yield an 85 kDa and a 25
kDa fragment. PARP cleavage is considered to be one of the classical
characteristics of apoptosis.
An anti-PARP-FITC conjugated Cleavage Site-Specific Antibody (CSSA) that
can detect apoptotic cells by flowcytometry. An alternative to the TUNEL
assay,
PARP Universal Colorimetric Assay measures the activity of PARP in cells
and tissues by detecting the incorporation of biotinylated Poly (ADP-ribose)
onto histone proteins.

ENDONUCLEASE ASSAY
Principle;
During apoptosis the endonuclease enxymes are activated to
Cleave the genomic DNA in to many smaller fragments
Steps
1. Preparation of agarose gel containing
Ethidium bromide and genomic DNA
2. Making well
3. Preparation of cell lysate
4. Incubation in humidified atmosphere
after loading cell lysate
5. Observe the DNA degradation

Detection of Caspase-3: Fluorometric Assay
Activation of proteases/caspases initiates apoptosis in mammalian cells.
The Caspase-3 Fluorometric Protease Assay provides a simple and
convenient means for assaying the DEVD-dependent caspase activity.
The assay is based on detection of cleavage of substrate DEVD-AFC (AFC:
7-amino-4-trifluoromethyl coumarin). DEVD-AFC emits blue light ( max =
400 nm); upon cleavage of the substrate by related caspases, free AFC
emits a yellow-green fluorescence ( max = 505 nm), which can be
quantified using a fluorometer or a fluorecence microtiter plate reader.
Comparison of the fluorescence of AFC from an apoptotic sample with an
un induced control allows determination of the fold increase in caspase-3
activity.
BioVision

COLORIMETRIC METHOD: CASPASE-3 DETECTION
The substrate, DEVD-pNA, is composed of the chromophore, p-nitroanilide (pNA), and a
synthetic tetrapeptide, DEVD (Asp-Glu-Val-Asp), which is the upstream amino acid
sequence of the Caspase-3 cleavage site in PARP. Upon cleavage of the substrate by
Caspase-3 or related caspases, free pNA light absorbance can be quantified using a
spectrophotometer or a microplate reader at 400 or 405 nm.
Comparison of the absorbance of pNA from apoptotic sample with an uninduced control
allows determination of the increase in Caspase-3 activity.
Bio Source

Mechanism of Colori
and flurimetric
detection of caspase -3

Fig. In Hoechst 33258 / PI double staining, cells with blue intact
nuclei were viable cells, whereas those with blue fragmented nuclei
were early apoptotic cells. Cells with pink intact nuclei were necrotic
cells, whereas cells with pink fragmented nuclei were late apoptotic
cells. (blue against Hoechst33258, red against PI)
Apoptotic(0%)
Necrotic(10.5%)
Apoptotic(85.2%)
Necrotic(11.2%)
Apoptotic(1.2%)
Necrotic(92.5%)

Clonogenic Cell:
 Defined as a cell with the capacity for sustained
proliferation
 Have undergone a minimum of 5-6 doublings to give
rise to colonies containing at least 50 cells
Colony-forming Efficiency

Colony-forming Efficiency (CFE)
 The ability to form colonies is used as a measure of
reproductive integrity
 It is often referred to as plating efficiency (PE)
Number of colonies formed
CFE =  100%
Number of cell plates
Clonogenic assays
 The basis of assays for determining the lethal effects of
cytotoxic agents

Determining the PE of an established
adherent cell line
Materials and Equipment
 Cell growth medium : Eagle’s basal medium (BME)
100 iu/ml penicillin,
0.1 mg/ml streptomycin
 Trypsin-EDTA
 Gentain violet stain

Procedure
1. Trypsinize monolayer cultures or use cell suspension
cultures and determine the viable cell count
2. Dilute cells in growth medium to 1000 , 2000 and
5000 cells/10ml
3. Inoculate nine replicate Petri dished with 4 ml growth medium plus
1ml cell suspension
4. Place plates in a humidified 5% CO2 plus air incubator are normal
growth temperature and rock shelf or tray gently to and fro three
times. The plates must not be moved now until colonies are stained
5. Stain and count three replicate per cell density at 1,2 and 3 weeks
(murine lines) or 2 , 3 and 4 weeks (human lined)
6. Calculate the optimum cell densities for seeding and duration of
incubation

Example; Rat keratinocytes
(A) (B)
(C) (D)
Colony forming Non-colony
forming
48 hr after
subculture
6 days after
subculture
: colony , : Single cells

Apoptosis assays detection and methods

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