Description
The caspase family of cysteine proteases plays a key role in apoptosis and inflammation. Caspase-3 is a key protease that is activated during the early stages of apoptosis and, like other members of the caspase family, is synthesized as an inactive pro-enzyme that is processed in cells undergoing apoptosis by self-proteolysis and/or cleavage by another protease. The processed forms of caspases consist of large (17-22 kDa) and small (10-12 kDa) subunits which associate to form an active enzyme. Active caspase-3, a marker for cells undergoing apoptosis, consists of a heterodimer of 17 and 12 kDa subunits which is derived from the 32 kDa pro-enzyme. Active caspase-3 proteolytically cleaves and activates other caspases, as well as relevant targets in the cytoplasm, e.g., D4-GDI and Bcl-2, and in the nucleus (e.g. PARP).This antibody has been reported to specifically recognize the active form of caspase-3 in human and mouse cells.It has not been reported to recognize the pro-enzyme form of caspase-3.
Components
| Part No. | Description | Size |
|---|---|---|
| 51-68654X | FITC Rabbit Anti- Active Caspase-3 | 100Tests (1 ea) |
| 51-6896KC | Cytofix/Cytoperm™ Fixation and Permeabilization Solution (1X) | 65mL (1 ea) |
| 51-6897KC | Perm/Wash™ Buffer (10X Solution) | 65mL (1 ea) |
| Resources & Tools | ||||||
|---|---|---|---|---|---|---|
| SpectrumViewer | Download MSDS | Download TDS | ||||
Preparation and Storage
Store undiluted at 4°C and protected from prolonged exposure to light.Do not freeze.
Product Notices
- 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).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
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).
Procedure
1. Add camptothecin (4-6 µM final concentration) to 1x10^6 /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 (1X) and antibody you will need so that each test will have 100 µl BD Perm/Wash™ buffer (1X) and 20 µl antibody (see chart).
2. Dilute the needed amount of BD Perm/Wash™ buffer (10X) 1:10 in distilled water prior to use.
Note: Precipitate may be occassionally observable 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 µm filter.
3. Wash cells twice with cold 1X PBS, then resuspend cells in BD Cytofix/Cytoperm™ solution at a concentration of 1x10^6 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 (1X) at a volume of 0.5 ml buffer/1x10e6 cells at room temperature.
6. Resuspend cells in the above calculated BD Perm/Wash™ buffer (1X) plus antibody and incubate for 30 min at room temperature.
7. 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.
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: Inhalative.
May cause respiratory irritation.
Precautionary statements
Wear protective clothing / eye protection.
Wear protective gloves.
Do not breathe mist/vapours/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.
