Cells were washed in PBS and stained in PBS with Live/Dead Fixable Blue Dead Cell Stain (Thermo Fisher Scientific) on ice for 25 min. Cells were washed with PBS/1% BSA and fixed with 2% formaldehyde (v/v) on ice for 15 min. The fixed cells were then washed with PBS/1% BSA and permeabilized with PBS/1% BSA supplemented with 0.03% saponin (PBS/1% BSA/0.03% saponin) on ice for 10 min. Fixed and permeabilized cells were then washed and incubated with anti-cleaved caspase-3 Alexa 647 (Cell Signaling) in PBS/1% BSA/0.03% saponin on ice for 30 min. Cells were washed, fixed in 1% formaldehyde (v/v), and analyzed on an LSRII (BD Biosciences).
Anti cleaved caspase 3 alexa 647
Manufactured by Cell Signaling Technology
Anti-cleaved caspase-3 Alexa 647 is a fluorescently-labeled primary antibody that specifically targets the cleaved form of caspase-3, a marker of apoptosis. This antibody is conjugated to the Alexa Fluor 647 dye, enabling fluorescent detection.
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2 protocols using anti cleaved caspase 3 alexa 647
1
Apoptosis Detection by Flow Cytometry
2
Multiparametric Flow Cytometry Analysis
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For surface staining, cells were washed in PBS and stained with Live/Dead Fixable Blue Dead Cell Stain (ThermoFisher Scientific) for 25 min on ice. Cells were washed and incubated for 25 min on ice with anti-CD25-BV421 (BioLegend, San Diego, CA, USA), anti-CD44-PE (BioLegend), anti CD4-PE-TexasRed (ThermoFisher Scientific), anti-CD8-PerCP-Cy5.5 (BioLegend), anti-TCRβ-PE-Cy7 (BioLegend), and anti-CD45RB-FITC (BioLegend). Cells were washed, fixed in 1% (v/v) methanol-free formaldehyde, and analyzed on an LSRII (BD Biosciences).
For intracellular staining, after staining with Live/Dead Fixable Blue Dead Cell Stain and surface antibodies as described above, cells were washed and fixed with 2% formaldehyde (v/v) for 15 min on ice. Fixed cells were washed and permeabilized with PBS/1% BSA supplemented with 0.03% saponin for 10 min on ice. Cells were washed and incubated with anti-cleaved caspase-3 Alexa 647 (Cell Signaling) for 30 min on ice. Cells were washed, fixed in 1% formaldehyde (v/v), and analyzed on an LSRII (BD Biosciences).
All flow cytometry data were analyzed with FlowJo v10 software (FlowJo, Ashland, OR, USA).
For intracellular staining, after staining with Live/Dead Fixable Blue Dead Cell Stain and surface antibodies as described above, cells were washed and fixed with 2% formaldehyde (v/v) for 15 min on ice. Fixed cells were washed and permeabilized with PBS/1% BSA supplemented with 0.03% saponin for 10 min on ice. Cells were washed and incubated with anti-cleaved caspase-3 Alexa 647 (Cell Signaling) for 30 min on ice. Cells were washed, fixed in 1% formaldehyde (v/v), and analyzed on an LSRII (BD Biosciences).
All flow cytometry data were analyzed with FlowJo v10 software (FlowJo, Ashland, OR, USA).
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