A RAPID METHOD FOR THE MEASUREMENT OF APOPTOSIS AND NECROSIS BY FLOW CYTOMETRY.
 
Huw S. Kruger Gray (1) and Robert Hsueh (2).
 
 
(1) Purdue University Cytometry Laboratories,
1515, Hansen Hall, B050,
West Lafayette, Indiana, 47907-1515, U.S.A.
tel: +1-765-494-0757.
fax: +1-765-494-0517.
E-mail: Zip@flowcyt.cyto.purdue.edu
 
(2) University of Texas, Southwestern Medical Centre,
Department of Pathology,
5323, Harry Hines Boulevard,
Dallas, Texas, 75235, U.S.A.
 

Summary.

A method is described for the rapid determination of cell-cycle phase, apoptosis and necrosis using flow cytometry. The procedure involves first staining cells for any surface markers, followed by staining with a membrane-impermeant DNA dye to label non-viable (necrotic) cells, then fixation, staining with a second DNA dye to label cycling and apoptotic cells, followed finally by analysis using multi-colour flow cytometry. The method is illustrated with examples of antibody-treated WEHI transfectant cells.

 


Determination of cell cycle phase, apoptosis and necrosis in WEHI-231 cells.

 
Materials and methods.

Examples are presented, of WEHI-231 cells containing a c-myc transfection which were treated with a goat anti-mouse IgM antibody for periods of 24 hours at an antibody concentration of 1ug./ml.; 24 hours at 25 ug./ml. and a control transfectant treated for 0 and 48hrs. at 1 ug./ml/. antibody. Data were analysed to observe for evidence of cell-cycle arrest, apoptosis and necrosis.

 
Staining procedure.

  1. Stain cells for surface markers (eg. using FITC- and/or PE-conjugated reagents), as desired.
  2. Wash cells once with 0.1% PBS/BSA/azide and decant supernatant.
  3. Add 50ul. of 400mM. 7-amino-actinomycin-D (7-AAD) (Sigma).
  4. Incubate for 30 minutes at 4 degrees Celcius.
  5. Without washing, add 1.0ul. of 0.5% paraformaldehyde in PBS.
  6. Add 220ul. of 0.01mg./ml. Hoechst 33342 (Molecular Probes).
  7. Incubate over-night at 4 degrees Celcius.
  8. Filter out any large clumps and/or debris.
  9. Run on the flow cytometer, using 488 nm. excitation to excite the FITC, PE (if appropriate) and 7-AAD fluorochromes, plus 350nm. (ie. U.V.) excitation to excite the Hoechst 33342 dye. Acquire the Hoechst fluorescence data using pulse-processing to obtain area and width measurements, for doublet descrimination purposes.
  10. Analyse the DNA data, after first gating out any cell doublets from cytograms of Hoechst fluorescence signal pulse area vs. width, as final cytograms showing Hoechst pulse area vs. 7-AAD pulse area.
 

Results and discussion.

These data were acquired using a multi-laser FACStar-Plus flow cytometer (Becton Dickinson), with an Hewlett-Packard 340 computer system running the Lysis-II software (Becton Dickinson) under the Consort-32 operating system. The data files were transfered subsequently across to a PC (Brysis) and re-analysed using the WinList software (Verity Software), then pasted in to Xara (Corel) for exportation as image files. The figures all were gated to exclude cell doublets and show cytograms of Hoechst fluorescence pulse area vs. 7-AAD pulse area.

Figure 1: This shows the control cell line, after treatment with no antibody for 48 hours. Normal cells can be seen in all phases of the cell cycle (G0/G1; S; G2/M), together with a very few cells showing some evidence of apoptosis and necrosis. Figure 2: This shows the control cell line, after treatment with 1 ug./ml. antibody for 48 hours. Some cells can be seen in the G0/G1 phases of the cell cycle, a few residual cells in S-phase and none evident in the G2/M phases. Many cells can be seen undergoing apoptosis and moving from there in to necrosis. Figure 3: This shows the c-myc transfected cell line, after treatment with 1 ug./ml. antibody for 24 hours. Viable cells can be seen in the G0/G1 phases of the cell cycle, with a much reduced number in the G2/M phases and some still undergoing DNA sysnthesis (ie. in the S-phase), together with many more cells showing evidence of apoptosis and necrosis. Cells can be seen also in the process of migrating from apoptosis to necrosis, as they die. Figure 4: This shows the c-myc transfected cell line, after treatment with 1 ug./ml. antibody for 48 hours. Viable cells can be seen in all phases of the cell cycle, plus a much reduced number of cells showing any evidence of apoptosis, with some others in necrosis. Figure 5: This shows the c-myc transfected cell line, after treatment with 25 ug./ml. antibody for 24 hours. Viable cells can be seen in the G0/G1 phases of the cell cycle, plus reduced numbers in S and G2/M phases, together with many cells showing evidence of apoptosis, necrosis and migration from apoptosis to necrosis.

The results above illustrate how this relatively swift and simple procedure can be used to observe changes in cell-cycling, entry in to programmed cell death (ie. apoptosis) and from there in to actual cell death (ie. necrosis), as a response to external factors. The initial cell staining with 7-AAD (propidium iodide could be employed as a possible alternative) is used to label the non-viable (ie. necrotic) cells, whose cell membrane has become compromised and thus rendered permeable to the dye. After permeabilisation by fixation with paraformaldehyde, the DNA of the viable cells (whose membrane was intact and thus their DNA has not been labelled by the 7-AAD) is stained with the second (Hoechst) DNA dye for analysis of cell cycle phase and apoptosis (where cells tend to lose fragments of degraded DNA and thus show a reduced DNA content, hence lower Hoechst fluorescence). The major draw-back to this method of analysis, is its requirement for a multi-laser flow cytometer with U.V. excitation capability.

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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 http://www.cyto.purdue.edu, EMAIL cdrom3@flowcyt.cyto.purdue.edu