APO-DIRECTTM PROTOCOL

The following protocol describes the method for measuring apoptosis in the positive and negative control cells that are provided in the APO-DIRECTTM kit. The same procedure should be employed for measuring apoptosis in the cell specimens provided by the researcher.

  1. Resuspend the positive (brown cap) and negative (natural cap) control cells by swirling the vials. Remove 1 ml aliquots of the control cell suspensions (approximately 1 x 106 cells per 1 ml) and place in 12 x 75 mm flow cytometry centrifuge tubes. Centrifuge (300 x g) the control cell suspensions for 5 minutes and remove the 70% (v/v) ethanol by aspiration being careful to not disturb the cell pellet.

  2. Resuspend each tube of control cells with 1 ml of Wash Buffer (blue cap) for each tube. Centrifuge as before and remove the supernatant by aspiration.

  3. Repeat the Wash Buffer treatment (step 2).

  4. Resuspend each tube of the control cell pellets in 50 µl of the Staining Solution (prepared as described below).

    Staining SOLUTION
    COMPONENT
    		1 ASSAY 6 ASSAYS
    (2 controls + 4 unknown)         		12 ASSAYS
    (2 controls + 10 unknown)
    TdT Reaction Buffer(green cap)
    TdT Enzyme(yellow cap)
    Fluorescein-dUTP(orange cap)
    Distilled H2O
         		10.00 µl
    0.75 µl
    8.00 µl
    32.00 µl
         		60.0 µl
    4.5 µl
    48.0 µl
    192.0 µl
         		120.0 µl
    9.0 µl
    96.0 µl
    384.0 µl
    Total Volume 50.75 µl
         		304.5 µl
         		609.0 µl

    The appropriate volume of Staining Solution to prepare for a variable number of assays is based upon multiples of the component volumes combined for 1 Assay. Mix only enough DNA Labeling Solution to complete the number of assays prepared per session. The DNA Labeling Solution is active for approximately 24 hours.

  5. Incubate the cells in the Staining Solution for 60 minutes at 37C in a temperature controlled bath. Shake cells every 15 min. to resuspend.

    NOTE: The Staining Reaction can also be carried out at 22-24C overnight for the control cells. For samples other than the control cells provided in the kit, incubation times at 37C may need to be adjusted to longer periods of time.

  6. At the end of the incubation time add 1.0 ml of Rinse Buffer (red cap) to each tube and centrifuge each tube (300 x g) for five minutes. Remove the supernatant by aspiration.

  7. Repeat the cell rinsing (as in step 6 ) with 1.0 ml of the Rinse Buffer (red cap), centrifuge and remove the supernatant by aspiration.

  8. Resuspend the cells pellet in 1.0 ml of the Propidium Iodide/RNase A solution (amber bottle).

  9. Incubate the cells in the dark for 30 minutes at room temperature.

  10. Analyze the cells in Propidium Iodide/RNase Solution by flow cytometry.

  11. Analyze the cells within 3 hours of staining.