Trypan blue exclusion was used to assess cell viability. The induction of apoptosis was quantified by fluorescence-activated cell sorting (FACS) on treated cells stained with annexin V. Briefly, cells were washed, resuspended with annexin V binding buffer, stained with fluorescein isothiocyanate (FITC)–conjugated annexin V (Roche, Mannheim, Germany) for 15 min at room temperature in the dark, and then washed and counterstained with propidium iodide (PI). The analysis was performed by a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) at a wavelength of 488 nm using Cell QuestPro Software (Beckman-Coulter, Fullerton, CA).
Fitc conjugated annexin 5
Manufactured by Roche
Sourced in United States, Germany
FITC)-conjugated annexin V is a fluorescently labeled protein that binds to phosphatidylserine, a phospholipid that is exposed on the cell surface during apoptosis. This product can be used to detect and quantify apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Cacl2, by Merck Group (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cell quest pro software, by Beckman Coulter (1 mentions)
Annexin 5, by Roche (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Modfit lt software, by Verity Software House (1 mentions)
Propidium iodide, by Roche (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
4 protocols using fitc conjugated annexin 5
1
Apoptosis Quantification by FACS
2
Cell Cycle and Apoptosis Analysis
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Cell cycle distribution changes were evaluated using the Acridine Orange (AO) technique as previously described [23 (link)]. The percentage of cells in G0, G1, S, and G2 M was determined by measuring simultaneously the DNA and RNA total cellular content. The percentage of apoptotic cells was measured based on the decreased stainability of apoptotic elements in DNA green fluorescence (sub-G0/1 peak on DNA frequency histograms) coupled with a higher RNA red fluorescence (which is common to chromatin condensation); cell debris was excluded from the analysis on the basis of their forward light scatter properties. Cell cycle distribution was analyzed using the ModFit LT software (Verity Software House, Topsham, ME).
Induction of apoptosis was also assessed by measuring Annexin V binding to externalized phosphatidylserine, as previously described [24 (link)]. Briefly, cells were washed twice with PBS and resuspended in binding buffer (10 mM Hepes/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, Sigma Chemical Co.). FITC-conjugated Annexin V (Roche Diagnostic Corp., Indianapolis, Indiana, USA) was added at a final concentration of 1 μg/mL. The mixture was incubated at room temperature for 15 min in the dark prior to flow cytometric analysis. Membrane integrity was simultaneously assessed by propidium iodide (PI, 0.25 μg/mL) exclusion.
Induction of apoptosis was also assessed by measuring Annexin V binding to externalized phosphatidylserine, as previously described [24 (link)]. Briefly, cells were washed twice with PBS and resuspended in binding buffer (10 mM Hepes/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, Sigma Chemical Co.). FITC-conjugated Annexin V (Roche Diagnostic Corp., Indianapolis, Indiana, USA) was added at a final concentration of 1 μg/mL. The mixture was incubated at room temperature for 15 min in the dark prior to flow cytometric analysis. Membrane integrity was simultaneously assessed by propidium iodide (PI, 0.25 μg/mL) exclusion.
3
Probing Apoptosis Pathways in Virus-Infected Cells
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To explore the role of PA-X in virus-induced cell apoptosis, MDCK and A549 cells were inoculated with r-CK10, CK-PAX3 or CK-PAX5 at a MOI of 1. At 12 and 24 h p.i., both inoculated and non-inoculated cells were trypsinized and washed three times with PBS. Cells were then stained with FITC-conjugated annexin V and propidium iodide (PI) (Roche, Mannheim, Germany) for 15 min at room temperature in the dark. The stained cells were then analyzed using a flow cytometer (BD, San Jose, USA). Apoptotic cells are those with high annexin V but low PI staining, whereas necrotic cells are highly stained with both two dyes.
4
Quantitative Assessment of Apoptosis
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Induction of apoptosis was assessed by measuring Annexin V binding to externalized phosphatidylserine, as previously described [72 (link)]. Briefly, cells were washed twice with PBS and resuspended in binding buffer 10 mM Hepes/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 (Sigma Chemical Co.). FITC conjugated Annexin V (Roche Diagnostic Corp.) was added at a final concentration of 1 μg/ml. The mixture was incubated at room temperature for 15 min in the dark. Stained cells were analyzed by flow cytometry using a Accuri C6 flow cytometer (Becton Dickinson), while simultaneously assessing membrane integrity by propidium iodide (PI) (0.25 μg/ml) exclusion. Samples were analyzed using the CFlow Plus software.
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