1st+Experiment+with+Caspase+Apoptosis+Kit

Flow Cytometry Part

Original Protocol//:// //Induction of Apoptosis by Camptothecin// >>>>> 2 uM -> 2 uL >>>>> 6 uM -> 6 uL //Active Caspase-3 Staining Protocol//
 * 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.
 * Camptothecin molecular weight: 348.4 g/mol
 * 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.
 * 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)
 * Incubate the cells for 4 hr at 37 C
 * 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.
 * Number of Tests: 5
 * 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)
 * 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
 * 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.