Cells were seeded at a density of 1 × 105 into 6-well plates and left to adhere overnight. Cells were then treated with either rhTNF-α, AT-IAP or both and returned to the incubator for 72 h. Whole culture medium was collected and pooled with the corresponding trypsinized cells before being pelleted via centrifugation at 1000 rpm. Cell pellets were resuspended in 100 μl of binding buffer and 5 μl of Annexin V antibody (Life Technologies, Paisley, UK) was added to each sample alongside 5 μl of propidium iodide (PI) stain (50 μg/ml). Samples were incubated in darkness for 15 min before adding 320 μl of binding buffer and analyzing on the EPICS XL flow cytometer (Beckman Coulter, Buckinghamshire, UK). Caspase 3/7 activity assays were performed according to manufacturer's instructions (Promega, UK).
Annexin 5 antibody
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Annexin V antibody is a laboratory reagent used for the detection and quantification of apoptosis, or programmed cell death, in cells. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is externalized on the surface of apoptotic cells. The Annexin V antibody binds to this exposed phosphatidylserine, allowing for the identification and measurement of apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Epics xl flow cytometer, by Beckman Coulter (1 mentions)
Caspase 3 7 activity assay, by Promega (1 mentions)
Apoptosis detection kit, by R&D Systems (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facsaria 3, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Rhtrail, by R&D Systems (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
6 protocols using annexin 5 antibody
1
Annexin V and Caspase 3/7 Assay
2
Annexin V and PI Apoptosis Assay
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U251 cells treated with either C1 or DMSO for 48 hours was analyzed by flow cytometry with Annexin V antibody and Propidium Iodide (Life technologies, NY) using the Apoptosis Detection Kit (R&D Systems, MN) according to the manufacturer’s instructions. Data was confirmed by 3 independent experiments.
3
Annexin V Apoptosis Assay for Pancreatic Cancer
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Annexin V staining was performed to determine the percentage of apoptotic cells. After treatment with gemcitabine and EPZ015666 or EPZ015938, the pancreatic cancer cells were washed with cold PBS, resuspended in Annexin V binding buffer (10mM HEPES, 140mM NaCl, and 2.5mM CaCl2, pH 7.4) with an appropriate amount of FITC-conjugated Annexin V antibody (Life Technologies #A13199), and incubated at room temperature (RT) for 15 minutes. After washing with binding buffer, the cells were resuspended in 2 μg/ml propidium iodide (PI) (Sigma) in PBS plus RNase, incubated at RT for 15 minutes in the dark, and then analyzed using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA).
4
Apoptosis Quantification in PC3 Cells
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PC3 cells were cultured as described earlier and harvested after treating the cells with 30, 60 and 120 µM of oroxyquinone. The harvested cells were washed once with PBS, followed by binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl and 2.5 mM CaCl2). The cells were suspended at a count of 1–5 × 106 cells/mL. 5 µL of Annexin V antibody (Invitrogen Cat No. 88-8005-72) was added to 100 µL of the cell suspension and incubated for 10 min in the dark. A further 2 mL of binding buffer was added to each tube and centrifuged at 400× g for 5 min in room temperature. The pellets were further resuspended in 200 µL 1X binding buffer and 5 µL of 500 µg/mL PI was added before analysis with a flow cytometer.
5
Multiparametric Cell Analysis Protocol
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Cell sorting was done on MoFlo (Beckman Coulter, High Wycombe, UK), FACSAria III (BD, Wokingham, UK), or Influx (BD) machines, and analysis performed on a BD LSR‐Fortessa, with 4′,6‐diamidino‐2‐phenylindole (DAPI) as a dead cell marker. Apoptosis analysis used Annexin‐V antibody (a13202 Fluor‐568, Invitrogen, Loughborough, UK) and DAPI as per manufacturers' instructions. Cell cycle analysis used Propidium Iodide staining of ethanol‐fixed cells.
6
TRAIL-induced Apoptosis Assay
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Cells were plated at a confluency of 1 × 106 cells/6010 mm2 surface. Twenty-four hours later, HA1ER-derived clone F12 and subclones 1F8 and 1C9 were challenged with recombinant human TRAIL (rhTRAIL, 1 μg/ml, R&D Systems, cat. #375-LT-010) for 4 h upon which cells were collected, washed once with 1x PBS, labelled with Annexin V antibody (Invitrogen, cat. # A35108) following manufacturer’s instructions and analysed by flow cytometry (BD LSRFortessa X-20 Cell Analyzer, BD Biosciences).
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