To measure caspase-3 activity in piceatannol-treated HL-60 cells, FITC-conjugated Monoclonal Active Caspase-3 Antibody Apoptosis Kit I (BD Pharmingen™, USA) was used, as described previously [82 ,83 ] Cells were exposed to piceatannol (IC90 = 14 µM) for 48 h. Cells not exposed to piceatannol were used as control. Following treatment, cells (5 × 105 cells per sample) were collected and stained with FITC-conjugated anti-active caspase-3 antibody according to the manufacturer’s protocol, and samples were analyzed by flow cytometry (Becton Dickinson FACScan, USA).
Fitc conjugated monoclonal active caspase 3 antibody apoptosis kit 1
Manufactured by BD
Sourced in United States
The FITC-conjugated Monoclonal Active Caspase-3 Antibody Apoptosis Kit I is a laboratory tool used to detect and quantify apoptosis, a programmed cell death process, in cell samples. The kit contains a FITC-labeled monoclonal antibody that specifically binds to the active form of caspase-3, a key enzyme involved in apoptosis. This allows for the identification and measurement of apoptotic cells through flow cytometry or fluorescence microscopy.
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4 protocols using fitc conjugated monoclonal active caspase 3 antibody apoptosis kit 1
1
Caspase-3 Activity in Piceatannol-Treated HL-60 Cells
2
Caspase-3 Activity Assay in HT22 Cells
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Caspase-3 activity was measured using FITC-conjugated Monoclonal Active Caspase-3 Antibody Apoptosis Kit I (BD Pharmingen, USA). HT22 cells were seeded in 6-well plates (2 × 105 cells per well) and allowed to attach for 24 h. Next, cells were incubated in the absence (control) or presence of 3-FMC for 24 h. Following incubation, cells were stained with FITC-conjugated anti-active caspase-3 antibody according to the manufacturer’s protocol and flow cytometric analyses were performed (Becton Dickinson FACSCalibur, USA).
3
Quantification of Active Caspase-3 by Flow Cytometry
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The detection of active caspase-3 was performed using the FITC-conjugated monoclonal active caspase-3 antibody apoptosis kit I (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. Briefly, cells were washed with ice-cold PBS and suspended in Cytofix/Cytoperm™ solution. Subsequent to incubation on ice for 20 min, the cells were pelleted, aspirated and then washed with wash buffer at room temperature. The cells were suspended in the wash buffer containing 5% (v/v) FITC-conjugated anti-active caspase-3 antibody for 40 min at room temperature in the dark. The cells were then washed with the wash buffer and analyzed using a Cell Lab Quanta™ SC flow cytometer.
4
Apoptosis Assay for NEFA-Treated Insulin Cells
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RINm5F insulin-producing cells were seeded at 1 x 10 6 cells on 6 cm tissue culture dishes and treated after 24 h with NEFAs. The cells were trypsinized after 24 h exposure to different NEFAs and stained for active caspases with the FITC-conjugated monoclonal active caspase-3 antibody apoptosis kit I (BD Biosciences Pharmingen, San Diego, CA). For the detection of caspase-8, caspase-9 or caspase-12 staining kits from PromoKine (Heidelberg, Germany) were used according to the manufacturer's protocols. The cells were analysed by flow cytometry at the FL-1 channel (488/527 nm) for active caspase-3, -9, and -12 or at the FL-2 channel (488/575 nm) for active caspase-8 with the CyFlowML cytometer (Partec, Münster, Germany). The portion of caspase-negative cells within the untreated cell population was gated and, on the basis of this gate, the portion of cells in which the caspases were activated after NEFA treatment could be determined.
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