APOPTOSIS or programmed cell death is marked by a series of characteristics including loss of cell volume, zeiosis, clumping of chromatin and nuclear fragmentation into apoptotic bodies. (For a review see REFS). There are several methods that can be used to quantitate APOPTOSIS by flow cytometry. One of the most simplest methods is to use propidium iodide to stain the DNA and look for the sub-diploid, or Ao, population of cells from a cell cycle profile.

The most commonly used dye for DNA content/cell cycle analysis is PROPIDIUM IODIDE (PI). The PI intercalates into the major groove of double-stranded DNA and produces a highly fluorescent adduct that can be excited at 488 nm with a broad emission centred around 600 nm. Since PI can also bind to double-stranded RNA, it is necessary to treat the cells with RNase for optimal DNA resolution. The excitation of PI at 488 nm facilitates its use on the benchtop cytomters. [ PI can also be excited in the U.V. (351-364 nm line from the argon laser) which should be considered when performing multicolour analysis on the multibeam cell sorters.]. Other flow cytometric based methods include the TUNEL assay, which measures DNA strand breaks and Annexin V binding, which detects relocation of membrane phosphatidyl serine from the intracellular surface to the extracellular surface. More recently, one mechanism which has consistently been implicated in apoptosis is CASPASE activity (cysteine proteases), typically CASPASE-3, which can be detected using a fluorogenic substrate kit. Microscopic examination and detection of DNA laddering by gel electrophoresis should be used to confirm the flow cytometric results.

A.

Protocol for assessing apoptosis by detection of sub-diploid population :
(from Daryzynkiewicz et al…..)
1. Harvest cells and prepare single cell suspension in buffer (e.g. PBS + 2% FBS; PBS + 0.1% BSA)
2. Wash cells X2 and resuspend at 1-2 x 106 cells/ml.
3. Aliquot 1 ml cells in a 15 ml polypropylene, V-bottomed tube and add 3 ml cold absolute ethanol. (The ethanol can be added forcibly by expelling from a pipette or dropwise while vortexing…determine the best method for each cell type to prevent clumping and cell loss.)
4. Fix cells for at least 1 hour at 4oC. (Cells may be stored in 70 % ethanol at -20 oC for several weeks prior to PI staining and flow cytometric analysis).
5. Wash cells X2 in PBS. (It may be necessary to centrifuge cells at a slightly higher "g" to pellet after ethanol fixation as the cells become floculent.)
6. Resuspend cells in 0.5 ml PBS and add 0.2-1.0 ml DNA extraction buffer.
7. Incubate at room temperature for 5 min and centrifuge.
8. Add 1 ml of propidium iodide staining solution to cell pellet and mix well. Add 50 ul of RNase A stock solution and incubate for 30 min at room temperature
9. Store samples at 4oC until analysed by flow cytometry.

REAGENTS:

DNA Extraction Buffer:
192 ml 0.2M Na2HPO4, 8 ml 0.1M citric acid; pH 7.8

Propidium Iodide Staining Solution:
3.8 mM sodium citrate, 50 ug/ml PI [Sigma, P 4170] in PBS.

RNase A stock solution:
10 mg/ml RNase A [Worthington Biochemicals, RASE LS005649, LS005650] (boiled for 5 min, aliquoted and stored frozen at -20oC).

B.

Protocol for assessing apoptosis by Annexin V binding :

1. Harvest cells, wash twice in PBS (4oC) and resuspend at a concentration of 1 x 106 cells/ml in Binding Buffer.
2. Aliquot cells (100 ul) into FACs tubes and add Annexin V and/or viability dye.
3. Mix gently and incubate for 15 min at room temperature in the dark
4. 4. Add 400 ul of Binding Buffer to each tube and analyze immediately by flow cytometry.

Recommended tubes:

(i) cells alone
(ii) cells + Annexin
(iii) cells + PI (or 7-AAD)
(iv) cells + Annexin + PI (or 7-AAD)

Annexin V is available in biotin, FITC and PE formats. When using Annexin-V-FITC, PI can be used as the viability marker (5 ul of a 50 ug/ml stock solution). When using Annexin-V-PE, 7-AAD may be the preferred viability marker (1 ug/ml final concentration) as there is less spectral overlap of PE and 7-AAD than PE and PI. However, 7-AAD is not as bright as PI. If care is observed when setting up the parameters on the cytometer, FITC, PE and PI can be combined effectively.

REAGENTS:

Annexin-V-FITC (PharMingen, 65874X)
Annexin-PE (PharMingen, 65875X)
Binding Buffer 10X, (0.1M HEPES/NaOH, pH7.4; 140mM NaCL; 25mM CaCl2), (PharMingen, 66121A)

Propidium Iodide (Sigma, P 4170)
7-AminoActinomycin D (Sigma, A 9400)

References:

C.

Protocol for assessing apoptosis by detection of DNA strand breaks by the TUNEL assay :

1. Wash cells in PBS and resuspend 2 x 106 cells in 0.5 ml PBS.
2. Fix cells by addition of 5 ml of 1% paraformaldehyde. Incubate on ice for 15 min.
3. Wash cells twice and resuspend in 0.5 ml PBS in polypropylene tubes.
4. Permeabilize cells by addition of 3 ml of ice-cold ethanol (70%) for at least 4 hr on ice.
[Cells may be stored for several weeks in 70% ethanol at -20 oC]
5. Centrifuge sample, wash in PBS and resuspend cells in 50 ul of TdT reaction solution:

10ul TdT reaction buffer, 2.0 ul BrdUTP stock solution, 15 U TdT, 5 ul CoCL2 (10 mM), 33.5 ul H20

6. Incubate cells for 40 min at 37oC with occasional mixing. (or room temp. overnight)
7. Add 1.5 ml rinsing buffer and centrifuge.
8. Add anti-BrdU-FITC antibody (approx. 100 ul) and incubate at room temp. for 1 hr.
9. Add 1ml of PI staining solution, incubate for at least 30 min at room temp. in the dark.
10. Analyse for FITC/PI fluorescence with appropriate controls for instrument setup.

The exact incubation times for fixation, permeabilization, TdT may vary according to cell type. It may be necessary to optimize these for specific cells and applications.

REAGENTS:

TdT Reaction Buffer (5X):
1M potassium or sodium cacodylate, 125mM TRIS-HCl, pH 6.6, 1.25mg/ml BSA. Store at -20oC.

Anti-BrdU-FITC Antibody Solution:
0.3 ug anti-BrdU-FITC antibody, 0.3% w/v Triton X-100, 1% BSA, PBS to 100 ul volume per test.

PI Staining Solution:
Propidium Iodide 5 ug/ml PI [Sigma, P 4170], RNase RNase A [Worthington Biochemicals, RASE LS005649, LS005650] (Dnase free) 200 ug/ml, PBS, pH 7.2-7.4.

Many vendors sell kits with pre-prepared buffers and reagents. Alternatively, individual reagents may be purchased separately. The above protocol is a general guide and individuaql kits may vary in the amounts of reagents used.

D.

Protocol for assessing apoptosis by detection of CASPASE-3 Activity :

Details of the CASPASE-3 assay kit can be found in the Pharmingen APOPTOSIS Instruction manual.

Cat. # 6632AK, 6632BK