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Protocols
APO-DIRECT™ Procedure
For Cat. No. 556381, see Technical Data Sheet for methods.
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1.
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Suspend the cells in 1% (w/v) paraformaldehyde in PBS (pH 7.4)
at a concentration of 1-2 x 106 cells/ml.
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2.
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Place the cell suspension on ice for 30-60 minutes.
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3.
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Centrifuge cells for 5 min at 300 x g and
discard the supernatant.
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4.
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Wash the cells in 5 ml of PBS, then pellet
the cells by centrifugation. Repeat the wash and centrifugation.
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5.
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Resuspend the cell pellet in the residual PBS in the tube by
gently vortexing the tube.
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6.
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Adjust the cell concentration to 1-2 x 106 cells/ml in 70% (v/v)
ice cold ethanol. Let cells stand for a minumum of 30 minutes
on ice or in the freezer. See note below.
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7.
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Let cells stand for a minimum of 30 min on
ice or in the freezer.
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Note: In some biological systems storage of the cells at -20°C
in 70% (v/v) ethanol for at least 12–18 hr prior to staining for apoptosis
detection yields the best results. Cells can be stored at -20°C for several
months before use.
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1.
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Resuspend the positive (brown cap) and negative (clear cap)
control cells by swirling the vials. Remove 1.0 ml aliquots of
the control cell suspensions (~1 x 106 cells/ml) and
place in 12 x 75 mm centrifuge tubes. Centrifuge the control cell
suspensions for 5 min at 300 x g and remove the 70% (v/v) ethanol
by aspiration, being careful to not disturb the cell pellet.
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2.
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Resuspend each tube of control cells with
1.0 ml of Wash Buffer (blue cap) for each tube. Centrifuge as before
and remove the supernatant by aspiration.
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3.
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Repeat the Wash Buffer treatment (step 2).
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4.
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Resuspend each tube of the control cell pellets in 50 µl
of the Staining Solution (prepared as described below).
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5.
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Incubate the cells in the Staining
Solution for 60 min at 37°C. The reaction can also be carried out at RT
overnight for the control cells. For test samples, the 60 min
incubation time at 37°C may need to be adjusted to longer periods of
time.
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6.
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At the end of the incubation time, add 1.0
ml of Rinse Buffer (red cap) to each tube and centrifuge each tube at
300 x g for 5 min. Remove the supernatant by aspiration.
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7.
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Repeat the cell rinsing with 1.0 ml of the Rinse Buffer, centrifuge
and remove the supernatant by aspiration.
Note: PI/Rnase treatment is not necessary if cell cycle is
not being analyzed (Proceed to step 11).
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8.
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Resuspend the cell pellet in 1.0 ml of the PI/RNase A solution
(amber bottle).
Note: If the cell density is low, decrease the amount of PI/RNase
A solution to 0.3 ml.
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9.
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Incubate the cells in the dark for 30 min
at RT.
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10.
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Analyze the cells in PI/RNase A solution
by flow cytometry.
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11.
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Analyze the cells by flow cytometry within
3 hr of staining. Cells may begin to deteriorate if left overnight before
analysis.
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