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.

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