For detection of apoptosis, 2 × 105 DLD1 and SW620 cells (EV and CRHR2+) were treated in 6-well plates with 0.1uM Ucn2 and 100ng/ml CH11. After 48h, floating cells were collected and pooled with the attached cells harvested by trypsinisation. Cells were fixed and permiabilized with CytoFix/CytoPerm (Pharmingen, San Diego, CA, USA) and stained with PE-conjugated anti-active caspase-3 antibody (Pharmingen) or the corresponding isotype control, according to manufacturer’s instructions. Samples were analyzed using a Becton Dickinson FACScan Flow cytometer (San Diego, CA). For determination of Fas surface expression, 2 × 105 cells from EV and CRHR2+ transduced CRC cell lines, treated with 0.1uM Ucn2 for 24 h, were detached using Accutase (Sigma) and stained with FITC-conjugated anti-CD95-FITC antibody (cloneDX2) (Miltenyl Biotech Inc, San Diego, CA), or the corresponding isotype control, according to manufacturer’s instructions. Cells were analyzed by flow cytometry as described above.
Pe conjugated anti active caspase 3 antibody
Manufactured by BD
Sourced in United States
The PE-conjugated anti-active caspase-3 antibody is a laboratory reagent used for the detection and analysis of active caspase-3, a key enzyme involved in the apoptosis (programmed cell death) process. The antibody is labeled with the fluorescent dye Phycoerythrin (PE), allowing for the visualization and quantification of active caspase-3 in biological samples through flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm, by BD (1 mentions)
Accutase, by Merck Group (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)
Apc conjugated anti brdu antibody, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Facscalibur analyzer, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
4 protocols using pe conjugated anti active caspase 3 antibody
1
Apoptosis and Fas Expression Analysis
2
Intracellular Caspase-3 Activation in T Cells
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For intracellular flow cytometric detection of activated caspase-3, T cells were allowed to migrate within the collagen gel matrix, or in medium, for up to 20 h. Apoptosis was induced in control cells by allowing them to migrate for 6 h within the collagen gel matrix, to which anti-FAS (clone APO-1-3) antibodies (Enzo Life Sciences) and Protein A (Sigma-Aldrich) were incorporated at 200 ng/ml. Cells were recovered from gel or medium after collagenase digestion, as described above. Cells were then fixed and permeabilized using the Cytofix/Cytoperm Fixation/Permeabilization kit (BD). Cells were incubated with a 1:10 dilution of PE-conjugated anti–active-caspase-3 antibody (BD) in Perm/Wash buffer for 30 min at 4°C. Stained cells were acquired on a FACSCanto, and percentage of positive cells was calculated using FlowJo analysis software.
3
Flow Cytometry Analysis of Apoptosis and Proliferation in Leukemic Cells
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Surface staining of tNGFR leukemic cells was performed with PE-conjugated anti-human CD271 antibody (BD Biosciences). Apoptosis assays were carried out as described [8 (link)], using PE-conjugated-anti-active Caspase-3 antibody (BD Biosciences). BrdU incorporation assays were performed as described [6 (link)], using APC-conjugated anti-BrdU antibody (BD Biosciences). Flow cytometry acquisitions were carried out on a FACSCalibur™ analyzer (BD Biosciences) equipped to detect 4 fluorescent parameters with the assistance of BD CellQuest Software (BD Biosciences) and data were analyzed with FlowJo Software (Tree Star). ICN1 leukemic cells were sorted on a FACSAria™III (BD Biosciences) cell sorter on the basis of tNGFR and/or GFP expression with the assistance of BD FACSDiva Software (BD Biosciences). Migration analyses were performed by videomicroscopy, as described [6 (link)].
4
CFSE-Based NK Cell Cytotoxicity Assay
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One day before assay, 2.5 × 106 target cells were labeled with 0.5 uM CellTrace CFSE and incubated at 37 °C overnight. The next day, CFSE-labeled target cells and NK-92 cells were cocultured at 1:1 effector-to-target ratio at 37 °C for 2 hours. After two washes with cold PBS, cells were fixed using BD Cytofix/Cytoperm solution (Cat # 51-6896KC BD Biosciences). After fixation, cells were washed twice with BD Perm/Wash buffer (Cat # 51-6897KC BD Biosciences) and incubated with PE-conjugated anti-active caspase-3 antibody (Cat # 51-68655X BD Biosciences) for 30 minutes at room temperature. The cells were then washed twice with BD Perm/Wash buffer and analyzed on BD Accuri™ C6 flow cytometer and FlowJo software.
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