Caspase Kit

2015-01-13

azim58 - Caspase Kit

Original Protocol: FITC Active Caspase 3 Apoptosis Kit

Active Caspase-3 Staining Protocol

Place some 1X PBS on ice.
Determine total amount of experimental samples (tests) and calculate

the amount of BD Perm/Wash buffer (1X) and antibody you will need so
that each test will have 100 uL BD Perm/Wash buffer (1X) and 20 uL
antibody.
o Number of Tests: 5
o Number of Cells = (Number of Tests)*1*10^6 = 5*1*10^6 cells =
5*10^6 cells (4*10^6 Jurkat cells and 1*10^6 3T3 cells)
o Perm/Wash Volume = (Number of Tests)*0.1 mL = 0.5 mL
- Note that more Perm/Wash 1X will be needed for all of the
different washes
@ Approximate amount needed = (0.5*2+0.1+1+0.5)*(Number of
Tests) = (0.5*2+0.1+1+0.5)*(5) = 13 mL
@ Maybe make 15-20 mL

o Antibody Volume = (Number of Tests)*20*uL = 5*20 uL = 100 uL

Dilute the needed amount of BD Perm/Wash buffer (10X) 1:10 in

distilled water prior to use. Note: Precipitate may be occasionally
observalbe with the BD Perm/Wash buffer (10X) which will not effect
performance of the buffer. The precipitate may be removed by filtering
the 1X solution through a 0.45 um filter

Detach adherent cells with trypsin if necessary. The cells may wash

off with PBS alone.

May want to transfer cells to the type of tube that will be used for

flow cytometry (12X75 mm round bottom).

May want to count the number of dead cells using trypan blue before

fixing the cells.

Wash cells twice with cold 1X PBS, then resuspend cells in BD

Cytofix/Cytoperm solution at a concentration of 1*10e6 cells/0.5 mL
o Cells may be washed by adding solution, centrifuging (1,000-4,000
rpm for 5 min), decanting supernatent (don't remove the cells!), and
resuspending.

Incubate cells for 20 min on ice.
Pellet cells, aspirate, and discard BD Cytofix/Cytoperm solution;

wash twice with BD Perm/Wash buffer (1X) at a volume of 0.5 mL
buffer/1*10e6 cells at room temperature

Resuspend cells in the above calculated BD Perm/Wash buffer (1X) (100

uL) plus antibody (20 uL) and incubate for 30 min at room temperature.
Make sure to keep the solutions in the dark so that light does not
damage the dye.

Wash each test in 1.0 mL BD Perm/Wash buffer (1X), then resuspend the

test in 1 mL BD Perm/Wash buffer (1X) and analyze by flow cytometry.
o Note that the original protocol says that you should resuspend in
a final volume of 500 uL, but 1 mL works better for the flow
cytometer.