Annexin V and propidium iodide (PI) double staining was used to evaluate the MDA-MB-468 cell apoptosis treated with free apatinib and PTX, apatinib (2 µmol/L), PTX (0.2 µmol/L) and the two drugs. In short, 1×106 MDA-MB-468 cells were seeded into a 6-well plate overnight. Cells were treated with various concentrations of drugs for 24 h respectively. The cells were then harvested and stained with 10 µg/mL Annexin V-fluorescein isothiocyanate (FITC)/PI (Pharmingen, CA, USA). Cells were stained with 50 µg/mL PI and 100 µg/mL RNase A and were protected from light. Samples were evaluated by flow cytometer and results were analyzed using the software Flow Jo v7.6.1 (https://www.flowjo.com/solutions/flowjo ).
Annexin 5 fluorescein isothiocyanate fitc pi
Manufactured by BD
Sourced in United States
Annexin V-fluorescein isothiocyanate (FITC)/PI is a laboratory reagent used for the detection and quantification of apoptotic cells. It is a combination of two fluorescent dyes, Annexin V-FITC and propidium iodide (PI), which bind to specific cellular components during the apoptotic process.
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Lab products found in correlation
Annexin 5 fitc apoptosis detection kit, by Abcam (1 mentions)
Facscan, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Hoechst pi, by Beyotime (1 mentions)
Anti bax, by Santa Cruz Biotechnology (1 mentions)
2 7 dichlorofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Solarbio (1 mentions)
Navios flow cytometer, by Beckman Coulter (1 mentions)
4 protocols using annexin 5 fluorescein isothiocyanate fitc pi
1
Apoptosis Assay of MDA-MB-468 Cells
2
Apoptosis Detection by Annexin V-FITC/PI
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Annexin V-fluorescein isothiocyanate (FITC)/PI (BD Biosciences, Franklin Lakes, NJ, USA) staining was performed to detect the apoptotic cells by assaying translocated phosphatidylserine (14 (link)). In brief, the cells were incubated in six-well plates at a density of 1×106 cells per culture well for 24 h. The cells were cultured for 48 h following infection with the various recombinant adenoviruses (100 MOI). The cells were harvested, washed once with PBS and resuspended in binding buffer. The cells were subsequently stained with FITC-labeled annexin V (Annexin V-FITC Apoptosis Detection kit; BioVision, Inc., Mountain View, CA, USA), according to the manufacturer’s instructions, with simultaneous dye exclusion of PI. The samples were analyzed by flow cytometry (FACScan; BD Biosciences). Untreated cells were used as controls.
3
ARPE-19 cell line viability assay
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ARPE‐19 cells were obtained from Shanghai Institute of Chinese Academy Cell Biology, SS‐31 and fetal bovine serum were obtained from Invitrogen, MTT was purchased from Solarbio, annexin V−fluorescein isothiocyanate (FITC)/PI were obtained from BD Biosciences, Hoechst‐PI was obtained from Beyotime Institute of Biotechnology, 2’,7’‐dichlorofluorescein diacetate (H2DCFDA) was purchased from Invitrogen, anti‐Bax and anti‐Bcl‐2 antibodies were purchased from Santa Cruz Biotechnology.
4
Apoptosis Detection by Annexin V/PI Staining
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Cells were treated with Olaparib/DMSO ± IR, as described above. Annexin V-fluorescein isothiocyanate (FITC)/PI (BD Biosciences, San Diego, CA) double staining method was used to detect cells undergoing early and late apoptosis. Briefly, at the end of treatments both adherent and floating cells were harvested and suspended at 1 × 106 cells/ml in 1X binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Then, 5 µl of Annexin V-FITC and 1 µl PI (final concentration 1 μg/ml) were added, and cells were incubated for 15 min in the dark. The double-stained cells were analyzed by flow cytometry using the Beckman Coulter Navios flow cytometer within 30 min from staining and data collected and analyzed using the FCS Express 7 software. The binding of Annexin V to cells with the integer cell membrane (i.e., in the absence of PI co-staining) was used as a marker of early apoptosis. The binding of Annexin V to cells with the non-integer cell membrane (i.e., positively co-staining for PI) was used as a marker of late apoptosis.
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