1st Experiment with Caspase Apoptosis Kit

2014-08-29

azim58 - 1st Experiment with Caspase Apoptosis Kit

Flow Cytometry Part

Original Protocol: FITC Active Caspase 3 Apoptosis Kit
Induction of Apoptosis by Camptothecin

Prepare a 1.0 mM stock solution of camptothecin (Sigma-Aldrich Cat.

No. C-9911) in DMSO. Camptothecin, an extract of the Chinese tree
Camptotheca acuminata, is a potent inhibitor of topoisomerase I, a
molecule required for DNA synthesis. Camptothecin has been reported to
induce apoptosis in a dose dependent manner in vitro.
o Camptothecin molecular weight: 348.4 g/mol
o 1 mmol/(1000 mL)* 1 mol/ (1000 mmol) * 348.4 g/(1 mol) * 1 mL =
3.484*10^(-4)g or 0.348 mg

Add camptothecin to 1X10e6/mL proliferating Cells. We will probably

use the max concentration of cells that we can fit in 3 mL.
Concentration of cells measured using trypan blue.
o Groups
- No camptothecin control
- 0.1 uM
- 2 uM
- 6 uM
- Jurkat (6 uM) (maybe. . . these cells weren't growing well.
@ Volume 1 mM stock needed
@ 1mM*y/(1000*uL)=0.0001 mM -> y=0.0001 mM * 1000 uL / (1
mM)
# 0.1 uM -> 0.1 uL (of 1 mM into 1 mL)
2 uM -> 2 uL
6 uM -> 6 uL

Incubate the cells for 4 hr at 37 C

Active Caspase-3 Staining Protocol

Detach adherent cells with trypsin if necessary
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

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

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) plus

antibody and incubate for 30 min at room temperature.

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

test in 0.5 mL BD Perm/Wash buffer (1X) and analyze by flow cytometry.