Description
Induction of Apoptosis by Camptothecin Materials 1. 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. 2. Jurkat cells (Human T-cell leukemia; ATCC® TIB-152™). Note: Mouse thymocytes or other cell types or cell lines can similarly be treated by culturing an agent that induces apoptosis such as Dexamethasone as shown. Procedure 1. Add camptothecin (4-6 µM final concentration) to 1 × 10^6 /ml proliferating Jurkat cells. 2. Incubate the cells for 4 hr at 37°C. Active Caspase-3 Staining Protocol Procedure 1. Determine total amount of experimental samples (tests) and calculate the amount of BD Perm/Wash™ buffer (1×) and antibody you will need so that each test will have 100 µl BD Perm/Wash™ buffer (1×) and 20 µl antibody as follows: No. Tests No. Cells Perm/Wash™ Vol. (ml) Antibody Vol. (μl) 1 1 × 10^6 0.1 20 5 5 × 10^6 0.5 100 10 10 × 10^6 1.0 200 20 20 × 10^6 2.0 400 2. Dilute the needed amount of BD Perm/Wash™ buffer (10×) 1:10 in distilled water prior to use. Note: Precipitate may be occasionally observable with the BD Perm/Wash™ buffer (10×) which will not affect performance of the buffer. The precipitate may be removed by filtering the 1× solution through a 0.45 µm filter. 3. Wash cells twice with cold 1× PBS, then resuspend cells in BD Cytofix/Cytoperm™ solution at a concentration of 1 × 10^6 cells/0.5 ml. 4. Incubate cells for 20 min on ice. 5. Pellet cells, aspirate, and discard BD Cytofix/Cytoperm™ solution; wash twice with BD Perm/Wash™ buffer (1×) at a volume of 0.5 ml buffer/1 × 10^6 cells at room temperature. 6. Resuspend cells in the above calculated BD Perm/Wash™ buffer (1×) plus antibody and incubate for 30 min at room temperature. 7. Wash each test in 1.0 ml BD Perm/Wash™ buffer (1×), then resuspend the test in 0.5 ml BD Perm/Wash™ buffer (1×) and analyze by flow cytometry. Danger: Cytofix/Cytoperm Fixation and Permeabilization Solution (1X) (component 51-6896KC) contains 4.2% formaldehyde. Hazard statements Harmful if inhaled. Causes skin irritation. Causes serious eye damage. May cause an allergic skin reaction. Suspected of causing genetic defects. May cause cancer. Route of exposure: Inhalation. May cause respiratory irritation. Precautionary statements Wear protective clothing / eye protection. Wear protective gloves. Do not breathe mist/vapors/spray. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. If skin irritation or rash occurs: Get medical advice/attention.
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions and unconjugated antibody and free dye were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
4. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
6. An isotype control should be used at the same concentration as the antibody of interest.
7. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
8. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
9. For U.S. patents that may apply, see bd.com/patents.
