Flow cytometric analysis of Active Caspase-3 expression by cells treated to induce apoptosis. Human Jurkat Cells - Cells from the Human Jurkat (T-cell leukemia, ATCC® TIB-152™) cell line were left Untreated or Treated (6 h) with 12 μM Camptothecin to induce apoptosis as indicated. Cells were washed, fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were subsequently stained with Purified Rabbit Anti-Active Caspase-3 antibody(Cat. No. 570524/570525) at 0.125 µg/test and secondarily stained with FITC Goat Anti-Rabbit IgG (FITC-Anti-Ig; Cat. No. 554020) at 0.06 µg/test. The fluorescence histograms showing Active Caspase-3 expression were derived from gated events with the light-scatter characteristics of single cells. Mouse Thymocytes - C57BL/6 Mouse thymocytes were left Untreated or treated (5 h) with 1 μM of Dexamethasone to induce apoptosis as indicated. The cells were similarly washed, fixed, permeabilized and stained as the Human Jurkat T cells. The fluorescence histograms showing Active Caspase-3 expression were derived from gated events with the light-scatter characteristics of single thymocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Analyses of Active Caspase-3 expression by Jurkat cells treated to induce apoptosis. Left Panel: Immunofluorescent Analysis: Jurkat cells (T-cell leukemia, ATCC® TIB-152™) were left Untreated (Left Image) or Treated (Right Image) with 6 µM Camptothecin for 4 hours to induce apoptosis. Cytospins of the Jurkat cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). and stained with Purified Rabbit Anti-Active Caspase-3 antibody (Cat. No. 570524/570525). After washing, cells were secondarily stained with BD Horizon™ BV480 Goat Anti-Rabbit IgG antibody (Cat. No. 564879; pseudocolored green) and with DRAQ7™ (cat. no. 564904, pseudocolored red) to stain nuclei. Active Caspase-3 staining was almost exclusively identified in cells treated to undergo apoptosis. Images acquired with 20x objective. Right Panel: Immunoprecipitation and Western blot analysis of Active Caspase-3 present in lysates from Human Jurkat cells treated to induce apoptosis. Cells from the Human Jurkat (T-cell leukemia, ATCC® TIB-152™) cell line were left Untreated (Left Panel) or Treated with 12 μM of Camptothecin for 6 hours to induce apoptosis (Right Panel). Immunoprecipitation: Cell lysates were immunoprecipitated with 2 or 4 µg/ml of Purified Rabbit Anti-Active Caspase-3 antibody (Cat. No. 570524/570525), Lanes 1 and 3 (4 µg) and lanes 2 and 4 (2 µg). Western blot: Active Caspase-3 was detected by Western blot analysis with an antibody that recognizes both the Pro-enzyme and Active Caspase-3 forms, reflecting the presence or absence of the caspase-3 pro-domain [lanes 1-4]. The results show that the Purified Rabbit Anti-Active Caspase-3 antibody immunoprecipitated only the active form of Caspase-3.
Flow cytometric analysis of Active Caspase-3 expression by cells treated to induce apoptosis. Human Jurkat Cells - Cells from the Human Jurkat (T-cell leukemia, ATCC® TIB-152™) cell line were left Untreated or Treated (6 h) with 12 μM Camptothecin to induce apoptosis as indicated. Cells were washed, fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were subsequently stained with Purified Rabbit Anti-Active Caspase-3 antibody(Cat. No. 570524/570525) at 0.125 µg/test and secondarily stained with FITC Goat Anti-Rabbit IgG (FITC-Anti-Ig; Cat. No. 554020) at 0.06 µg/test. The fluorescence histograms showing Active Caspase-3 expression were derived from gated events with the light-scatter characteristics of single cells. Mouse Thymocytes - C57BL/6 Mouse thymocytes were left Untreated or treated (5 h) with 1 μM of Dexamethasone to induce apoptosis as indicated. The cells were similarly washed, fixed, permeabilized and stained as the Human Jurkat T cells. The fluorescence histograms showing Active Caspase-3 expression were derived from gated events with the light-scatter characteristics of single thymocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
Analyses of Active Caspase-3 expression by Jurkat cells treated to induce apoptosis. Left Panel: Immunofluorescent Analysis: Jurkat cells (T-cell leukemia, ATCC® TIB-152™) were left Untreated (Left Image) or Treated (Right Image) with 6 µM Camptothecin for 4 hours to induce apoptosis. Cytospins of the Jurkat cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). and stained with Purified Rabbit Anti-Active Caspase-3 antibody (Cat. No. 570524/570525). After washing, cells were secondarily stained with BD Horizon™ BV480 Goat Anti-Rabbit IgG antibody (Cat. No. 564879; pseudocolored green) and with DRAQ7™ (cat. no. 564904, pseudocolored red) to stain nuclei. Active Caspase-3 staining was almost exclusively identified in cells treated to undergo apoptosis. Images acquired with 20x objective. Right Panel: Immunoprecipitation and Western blot analysis of Active Caspase-3 present in lysates from Human Jurkat cells treated to induce apoptosis. Cells from the Human Jurkat (T-cell leukemia, ATCC® TIB-152™) cell line were left Untreated (Left Panel) or Treated with 12 μM of Camptothecin for 6 hours to induce apoptosis (Right Panel). Immunoprecipitation: Cell lysates were immunoprecipitated with 2 or 4 µg/ml of Purified Rabbit Anti-Active Caspase-3 antibody (Cat. No. 570524/570525), Lanes 1 and 3 (4 µg) and lanes 2 and 4 (2 µg). Western blot: Active Caspase-3 was detected by Western blot analysis with an antibody that recognizes both the Pro-enzyme and Active Caspase-3 forms, reflecting the presence or absence of the caspase-3 pro-domain [lanes 1-4]. The results show that the Purified Rabbit Anti-Active Caspase-3 antibody immunoprecipitated only the active form of Caspase-3.
Product Details
BD Pharmingen™
Apopain; CASP-3; CASP3; CC3; CPP-32; CPP32; Caspase 3; Yama
Human (QC Testing), Mouse (Tested in Development)
Rabbit IgG, κ
Human Active Caspase-3 Fragment
Intracellular staining (flow cytometry) (Routinely Tested), Immunofluorescence, Immunoprecipitation (Tested During Development)
0.5 mg/ml
836, 12367
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- For U.S. patents that may apply, see bd.com/patents.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
FITC Goat Anti-Rabbit IgG RUO
Size
0.5 mg
Cat No.
554020
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DRAQ7™ RUO
Size
1 mL
Cat No.
564904
570524 Rev. 2
Antibody Details
C92-605.rMAb
The C92-605.rMAb is a recombinant monoclonal antibody derived from C92-605 hybridoma cells. This antibody specifically recognizes 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. The Caspase family of cysteine proteases play crucial roles 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, eg, D4-GDI and Bcl-2, and in the nucleus (eg, PARP).
570524 Rev. 2
Format Details
Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
570524 Rev.2
Citations & References
Development References (5)
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Dukers DF, Oudejans JJ, Vos W, ten Berge RL, Meijer CJ. Apoptosis in B-cell lymphomas and reactive lymphoid tissues always involves activation of caspase 3 as determined by a new in situ detection method. J Pathol. 2002; 196(3):307-315. (Clone-specific: Immunohistochemistry, Immunoprecipitation). View Reference
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Ohsawa S, Hamada S, Yoshida H, Miura M. Caspase-mediated changes in histone H1 in early apoptosis: prolonged caspase activation in developing olfactory sensory neurons. Cell Death Differ. 2008; 15(9):1429-1439. (Clone-specific: Fluorescence microscopy, Immunofluorescence, Western blot). View Reference
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Pettersen RD, Bernard G, Olafsen MK, Pourtein M, Lie SO. CD99 signals caspase-independent T cell death. J Immunol. 2001; 166(8):4931-4942. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Winzler C, Fantinato M, Giordan M, Calore E, Basso G, Messina C. CD4(+) T regulatory cells are more resistant to DNA damage compared to CD4(+) T effector cells as revealed by flow cytometric analysis.. Cytometry A. 2011; 79(11):903-11. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Zhu S, Zhang X, Weichert-Leahey N, et al. LMO1 Synergizes with MYCN to Promote Neuroblastoma Initiation and Metastasis.. Cancer Cell. 2017; 32(3):310-323.e5. (Clone-specific: Immunohistochemistry). View Reference
570524 Rev. 2
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
