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APOPTOSIS I
[adapted from: MA Hotz, J Gong, F Traganos, and Z Darzynkiewicz.
1994. Flow cytometric detection of apoptosis: Comparison of the
assays of in situ DNA degradation and chromatin changes. Cytom
15:237-44.]
Requires:
HBSS = Hanks balanced salt solution
CAM = camptothecin, DNA topoisomerase I inhibitor, 5 mg/ml in DMSO
(stock) [as positive control]
fix = 1 ml HBSS/9 mls 70% ETOH
SC Buffer = 0.05 M Na2HP04 (9 parts)/25 mM citric acid (1 part)/0.1 %
Triton X-100, pH 7.8
PI Buffer - 10 mM PIPES buffer(Calbiochem)/0.1 N NaCl, 2 mM MgCl2/0.1
% Triton X-100, pH 6.8 with 20 mg PI/50 U-ml-1 RNase A
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1. For positive apoptotic cells: incubate cells with 0.15 mM CAM
for up to 3 h.
2. trypsinize quickly, add cold media, centifuge quickly at 4 C
3. fix (1x10 7 cells/10 ml), 24-48 h, -20 0C
4. pellet, 200 g, 5 min
5. resuspend in 1 ml SC Buffer
6. stain by adding .2 ml SC suspension to 1.5 ml PI Buf, incubate
(at least) 30 min, in dark
Apoptosis II (alternative):
[adapted from: WG Telford, LE King, PE Fraker. 1992. Comparative
evaluation of several DNA binding dyes in the detection of
apoptosis-associated chromatin degradation by flow cytometry. Cytom
13:137-43.]
Requires:
PI - 0.1 % Triton X-100//0.1 mN EDTA(NA)2//50 mg/ml RNase A (50
U/mg)//50 mg/ml PI
1. fix 2x106 cells, chilled to 40C, rapidly resuspending in 80 %
ETOH, 4 0C.
2. incubate on ice 30 min
3. pellet 1200 rpm, 5 min
4. wash 1x PBS, pellet 1200 rpm, 5 min
5. resuspend in PI, in dark, analyze within 24 h
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web