Cells were seeded at 200,000 cells per well of 6 well dishes on the day prior to treatment and adhered overnight. For time course experiments, drugs were added in a manner such that all samples were collected at the same time. Post treatment, cells were collected and fixed with 4% formaldehyde for 15 minutes. After two washes with PBS, cells were exposed to 100% methanol at −20°C for >2 hours. Cells were washed with PBS and incubated with the active caspase-3 antibody (1:500 dilution, BD Biosciences) in a 50/50 (v/v) PBS-T:Odyssey blocking buffer solution (LICOR). Cells were washed with PBS-T and incubated with the Alexa-fluor 647 cleaved PARP primary and Alexa-fluor 488 goat anti-rabbit secondary antibodies (1:500 dilution, BD Bioscience) overnight at room temperature. FACS samples were run on an LSR II machine with excitation lasers of 488 and 640 nm.
Active caspase 3 antibody
Manufactured by BD
The Active caspase-3 antibody is a laboratory reagent used for the detection and quantification of active caspase-3, a key enzyme involved in the apoptosis (programmed cell death) process. This antibody specifically recognizes the cleaved, active form of caspase-3, providing a reliable tool for researchers studying cell death mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Pbs t odyssey blocking buffer solution, by LI COR (2 mentions)
Alexa fluor 647 cleaved parp primary and alexa fluor 488 goat anti rabbit secondary antibodies, by BD (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Fitc rabbit anti active caspase 3 antibody, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Alexa fluor 568 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Recombinant human granzyme b, by Enzo Life Sciences (1 mentions)
Bioporter quikease protein delivery kit, by Merck Group (1 mentions)
Live dead stain, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
7 protocols using active caspase 3 antibody
1
Apoptosis Assessment by Flow Cytometry
2
Quantifying Apoptosis via Caspase-3 Activity
3
Histological and Immunofluorescence Analysis of Zebrafish Embryos
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For histological analysis, zebrafish embryos at 48 hpf and 72 hpf were fixed overnight in 4% PFA at 4 °C, dehydrated with a graded ethanol, processed in xylene and embedded in paraffin. Cross section (6 μm) were stained with hematoxylin and eosin using a standard protocol44 (link). For whole mount immunofluorescence staining, zebrafish embryos at 48 hpf were fixed overnight in 4% PFA and dehydrated with methanol. Zebrafish embryos were permeabilized in acetone for 7 min at −20 °C and washed in water, followed by several washes in PBST. After blocking for 30 min in 2% horse serum, zebrafish embryos were incubated with active caspase-3 antibody (BD Biosciences) or p-histone H3 antibody (Millipore) at 4 °C overnight. On the next day zebrafish embryos were incubated with Alexa Fluor 568-conjugated secondary antibody (Invitrogen). Cryosection was performed as described previously45 (link).
4
Granzyme B-Mediated Apoptosis Induction
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Recombinant human granzyme B (Enzo Life Sciences, New York, NY, USA) was mixed with BioPORTER QuikEase Protein Delivery Kit (Sigma-Aldrich, St. Louis, MO, USA). 1 × 105 cells per well were plated in 12-well plates and cultured overnight at 37 °C. Cells were washed, and 100 ng of granzyme B in Opti-MEM (Thermo Fisher Scientific) was added to each well. After incubation for 4 h at 37 °C, the frequency of apoptotic cells was determined by staining with active caspase-3 antibody (BD Biosciences) and examined by flow cytometry as shown gating strategy in Supplementary Figure S1 .
5
Apoptosis Assay for Single Cells
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Organoids were dissociated into single cells with TrypLE Express (37 °C, 5 min) and cells were collected by centrifugation (2000rpm, 2 min). Cells were incubated in PBS with LIVE/DEAD stain (ThermoFisher) (10 min on ice), washed in FACS buffer (1% FBS in PBS), fixed in 0.5% PFA (37 °C, 15 min), permeabilised in 70% ethanol (10 min on ice), washed in FACS buffer, stained with Active Caspase-3 antibody (BD 560626) (30 min), washed and then resuspended in FACS buffer. Cells were analysed using LSRFortessa Cell Analyzer (BD Biosciences) and data analysed in FlowJo.
6
Apoptosis Assessment by Flow Cytometry
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Cells were seeded at 200,000 cells per well of 6 well dishes on the day prior to treatment and adhered overnight. For time course experiments, drugs were added in a manner such that all samples were collected at the same time. Post treatment, cells were collected and fixed with 4% formaldehyde for 15 minutes. After two washes with PBS, cells were exposed to 100% methanol at −20°C for >2 hours. Cells were washed with PBS and incubated with the active caspase-3 antibody (1:500 dilution, BD Biosciences) in a 50/50 (v/v) PBS-T:Odyssey blocking buffer solution (LICOR). Cells were washed with PBS-T and incubated with the Alexa-fluor 647 cleaved PARP primary and Alexa-fluor 488 goat anti-rabbit secondary antibodies (1:500 dilution, BD Bioscience) overnight at room temperature. FACS samples were run on an LSR II machine with excitation lasers of 488 and 640 nm.
7
Caspase-3 Activation Analysis
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At 24 hpf, embryos were binned based on genotypic differences in morphology and processed into single-cell suspensions, as described above. After the first spin at 900 g , the pellet was resuspended in 100 µl 4% PFA and fixed for 15 min at room temperature. Cells were washed with 1× D-PBS and permeabilized in a saponin-based wash buffer (0.1% saponin/1% BSA/D-PBS) for 15 min at room temperature. Cells were incubated with active caspase-3 antibody (BD Biosciences) at a 1:500 dilution in saponin-based wash buffer for 30 min at room temperature. Secondary antibody incubations were performed at 1:1000 (goat anti-rabbit AlexaFluor-594) for 30 min in the dark. Cells were resuspended in wash buffer and analyzed by flow cytometry using a BD LSRII-Yellow Analyzer. Quantitations were made using FlowJo 10.3 beta software.
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