Viable Hoechst 33342/SYTO 11/PI/CEN Protocol for Apoptosis

Ref. Syto - Poot, M. Gibson, LL, Singer, VL (1997) Detection of apoptosis in live cells by MitoTracker Red CMXRos and SYTO dye flow cytometry. Cytometry 27:358-364 PI Ð (really Ho/FDA/PI) Stohr M and Vogt-Schaden M. A new dual staining technique for simultaneous flow cytometric DNA analysis of living and dead cells IN Laerum, OD, Lindmo T. Thorud E. (eds) "Flow Cytometry" Vol IV Bergen; Norrway Universitetsforlaget pp 96-99 (1979) Hamori, E., Arndt-Jovin, D.J. Grimwade, B.G. and Jovin, T.M. Selection of viable cells with known DNA content. Cytometry 1:132-135. (1980). Poot M., Gollahon, K.A. Gollahon, P.S. Rabinovitch (1999) Werner syndrome lymphoblastoid cells are sensitive to camptothecin-induced apoptosis in S-phase, Human Genetics 104, 10-14.

This protocol determines the absolute number of live (Hoechst 33342 positive/PI negative, early apoptotic (Hoechst 33342 positive/PI negative, SYTO 11 low), dead (PI positive) cells and debris signals. Thus, one can follow cells through various stages of apoptosis and decay.

Make the following stock solutions:
  1. 1 mM Hoechst 33342 in distilled water (do NOT use PBS, since phosphates will precipitate the dye)
  2. 10 µM SYTO 11 dye (add 2 µL of the 5 mM stock solution to 1 mL distilled water)
  3. 1 mg/mL propidium iodide (Sigma) in distilled water.

The Hoechst 33342 and propidium iodide solutions keep in the refrigerator in the dark for weeks; the SYTO 11 has to be diluted on the same day as being used. CEN are chicken erythrocyte nuclei (named CEN Singlet Cytometry Control from Riese Enterprises, Inc. Grass Vly, CA)

  1. Bring cells into suspension; preferably at a density of 0.2 to 0.5 million per mL.
  2. Add sequentially per mL cell suspension:
    10 µL Hoechst 33342 (Molecular Probes cat # H-1399)
    10 µL SYTO 11 (Molecular Probes cat # S-7573)
    5 µL propidium iodide (Molecular Probes cat # P-1304)
    1 µL CEN (CEN Singlet Cytometry Control; Riese Enterprises, Inc. Grass Vly, CA)
  3. Incubate at 37° C for 30 minute
  4. Analyze on the flow cytometer using the Z-WRN-Toxicity protocol. Cells should be analyzed as soon after staining as possible.

Processing data by the Hoechst 33342/SYTO 11/PI/CEN Protocol for Apoptosis

This protocol determines the absolute number of live (Hoechst 33342 positive/PI negative, early apoptotic (Hoechst 33342 positive/PI negative, SYTO 11 low), dead (PI positive) cells and debris signals. Thus, one can follow cells through various stages of apoptosis and decay.

  1. Open your data files with MPLUS (in case of difficulties with this step ask Mike Shen for assistance).
  2. lick on GATE 2D (blue field on the bottom of the base page).
  3. Select PMT2 and PMT PK (red options on the GATE 2D page). This will select the PMT2 data (in this assay intensities of Hoechst 33342 fluorescence signals) on the X-axis and the PMT2 PK (peak height of the Hoechst 33342 fluorescence signals) on the Y-axis. A picture similar to the one shown in the top panel of the figure should appear.
  4. Draw a diagonal as shown in the figure. Single cells will appear above the diagonal, while clumps will appear below the diagonal. This is because clumps have a relatively lower peak height than do single particles of the same total fluorescence intensity.
  5. Click DONE.
  6. elect PMT 2 LOG and PMT 5 LOG. This selects the Log distribution of the Hoechst fluorescence signals of all cells on the X-axis and the Log distribution of the SYTO 11 and PI fluorescence signals of all cells on the Y-axis. The green fluorescence of SYTO 11 'bleeds' into the channel of the red PI fluorescence (PMT 5). In this way 'normal' cells (high SYTO 11 and PI negative) appear in the middle of the screen, 'apoptotic' cells (low SYTO 11 and PI negative) appear in the bottom half of the screen and 'dead' cells (PI positive) appear on the top of the screen.
  7. Draw the regions as indicated in the bottom panel of the figure and save the result by clicking on WINPRINT. This saves the image and the signal numbers within each frame.
  8. After you are done with all your data files, you can exit MPLUS by clicking 'exit' and use PRINT ALL to print all saved files.

Gating to exclude clumps

clump gate


Gating of "Normal", Apoptotic and Dead Cells and Debris in the Hoechst/SYTO 11/PI/CEN Assay

analysis gate