For proliferation assays, 5000 cells were plated in 96 well plates and incubated with recombinant TRAIL the following day for 24 h. Proliferation was analyzed with the Cell Proliferation Kit II (Roche) according to the manufacturer’s instructions and survival was calculated by normalizing treated to untreated cells. For apoptosis assays, 40,000 cells were plated in 24 well plates and incubated with recombinant TRAIL for 24 h. Cells were harvested with Trypsin and stained with Annexin V-488 (Biolegend) and propidium-iodide (Sigma) in Annexin binding buffer (10mM HEPES, 140mM NaCl, and 2.5mM CaCl 2, pH 7.4) for 15 min at RT. FACS analysis was performed on a FACScan instrument (BD) and cells negative for Annexin V and propidium iodide considered as alive. For colony forming assays, the indicated numbers of cells were plated in 12 well plates and grown for 7 days in medium with TRAIL changed every second day. Colonies were visualized by staining with 0.02% crystal violet (Sigma) in 50% methanol.
Annexin v 488
Manufactured by BioLegend
Annexin V-488 is a fluorescent conjugate of the protein Annexin V. Annexin V binds to phosphatidylserine, a phospholipid that is exposed on the surface of cells undergoing apoptosis. The Annexin V-488 conjugate can be used to detect and quantify apoptotic cells through flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (3 mentions)
Crystal violet, by Merck Group (2 mentions)
Propidium iodide, by BD (2 mentions)
Facscan, by BD (2 mentions)
Annexin 5, by BD (2 mentions)
Cell proliferation kit 2, by Roche (2 mentions)
α gapdh, by Cell Signaling Technology (1 mentions)
α flag m2, by Merck Group (1 mentions)
α γh2ax, by Cell Signaling Technology (1 mentions)
7 aad, by BioLegend (1 mentions)
Goat α mouse hrp, by Agilent Technologies (1 mentions)
α rabbit alexafluor 488, by Thermo Fisher Scientific (1 mentions)
P chk1 ser345, by Cell Signaling Technology (1 mentions)
α iκbα, by Cell Signaling Technology (1 mentions)
Nutlin 3, by Merck Group (1 mentions)
Annexin 5 apc, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Metafectene, by Biontex (1 mentions)
3 protocols using annexin v 488
1
TRAIL-induced Proliferation and Apoptosis
2
TRAIL-induced Proliferation and Apoptosis
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For proliferation assays, 5000 cells were plated in 96 well plates and incubated with recombinant TRAIL the following day for 24 h. Proliferation was analyzed with the Cell Proliferation Kit II (Roche) according to the manufacturer’s instructions and survival was calculated by normalizing treated to untreated cells. For apoptosis assays, 40,000 cells were plated in 24 well plates and incubated with recombinant TRAIL for 24 h. Cells were harvested with Trypsin and stained with Annexin V-488 (Biolegend) and propidium-iodide (Sigma) in Annexin binding buffer (10mM HEPES, 140mM NaCl, and 2.5mM CaCl 2, pH 7.4) for 15 min at RT. FACS analysis was performed on a FACScan instrument (BD) and cells negative for Annexin V and propidium iodide considered as alive. For colony forming assays, the indicated numbers of cells were plated in 12 well plates and grown for 7 days in medium with TRAIL changed every second day. Colonies were visualized by staining with 0.02% crystal violet (Sigma) in 50% methanol.
3
Apoptosis Induction Assay Protocol
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Chemicals and cytokines used were staurosporine, cycloheximide, doxorubicin, DAPI, H2O2, Nutlin-3, polybrene, propidium-iodide, doxocycline, puromycin (all Sigma), AnnexinV–APC, AnnexinV-488, 7-AAD (Biolegend), etoposide, camptothecin (Alexis). Antibodies: α-IκBα (#9242), α-PARP1 (#9532), α-GAPDH (#2118), α-Histone H3 (Ser10) (#9701), α-γH2A.X (#2577), pChk1 Ser345 (#2341), Chk1 (#2345) (Cell Signaling); α-p53 (sc-6243), α-tubulin (sc-32293) (Santa Cruz Biotechnology); α-PDCD5 (Proteintech); α-LaminB1 (gift from Peter Gruber); α-Flag (M2) (Sigma), α-p21 (#554262) (BD Pharmingen); α-rabbit AlexaFluor 488 (Invitrogen), goat α-rabbit HRP, goat α-mouse HRP (Dako). SiRNA targeting human PDCD5 (5′-CUAAAGCAGUAGAGAAUUATT-3′) was synthesised by Microsynth and transfected into 293T cells using Metafectene (Biontex) according to the manufacturer′s instructions.
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