Effect of miR-9 on OVCAR-3 tumor cell apoptosis was quantified by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC) cell apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol (18 (link)). Briefly, the OVCAR-3 cells were transfected with miR-9 or NC vectors for 36 h, followed by the replacement of cell culture medium with serum-free RPMI 1640 medium. Total cells were harvested and washed using PBS buffer (pH 7.4) three times, and resuspended in the staining buffer provided in the kit. Following this, 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) were mixed with the cells. After being cultivated at room temperature for 10 min, mixtures were analyzed using the FACScan flow cytometry (BD Biosciences). Annexin V-positive and PI-negative cells were considered to be apoptotic cells.
Annexin 5 fluorescein isothiocyanate fitc cell apoptosis kit
Manufactured by Thermo Fisher Scientific
Sourced in China
The Annexin V-fluorescein isothiocyanate (FITC) cell apoptosis kit is a laboratory equipment used to detect and quantify the extent of apoptosis, or programmed cell death, in cell populations. The kit utilizes the binding of Annexin V, a protein that has a high affinity for phosphatidylserine, to the cell membrane as an indicator of apoptosis. The FITC fluorescent label allows for the detection and analysis of Annexin V-positive cells using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Facscan flow cytometry, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Beyoclick edu 594 cell proliferation kit, by Beyotime (1 mentions)
3 methyladenine, by Selleck Chemicals (1 mentions)
Modified eagle s medium mem, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Epacadostat, by Selleck Chemicals (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Cellquest software, by BD (1 mentions)
Facscalibur flow cytometry, by BD (1 mentions)
4 protocols using annexin 5 fluorescein isothiocyanate fitc cell apoptosis kit
1
Quantifying miR-9's Impact on OVCAR-3 Apoptosis
2
Pharmacological Modulation of Cellular Pathways
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Curdione was purchased from Solarbio Science&Technology Co. Ltd. (Beijing, China). Modified Eagle’s medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Non-Essential Amino Acids (NEAA), Pymvate Sodium (NaP), and penicillin–streptomycin (PS) were purchased from Gibco (Waltham, MA, USA). The IDO1 inhibitor epacadostat was purchased from Selleck (S7910, Texas, USA) and autophagy inhibitor 3- Methyladenine was purchased from Selleck (S2767, Texas, USA), CCk8 was purchased from Dojindo (CK04, Kumamoto, Japan), Beyo Click™ Edu-594 Cell Proliferation Kit was purchased from Beyotime (C0078S, Shanghai, China), and Annexin V-fluorescein isothiocyanate (FITC) cell apoptosis kit was purchased from Invitrogen (V13241, New York, California, USA).
3
Flow Cytometric Apoptosis Assay
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Cell apoptosis was assayed by flow cytometry using an annexin V-fluorescein isothiocyanate (FITC) cell apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Briefly, after transfection for 48 h, cells were cultured with fresh serum-free DMEM/F12 medium at 37°C for 12 h. Subsequently, cells were harvested and washed three times (5 min/wash) with phosphate-buffered saline (pH 7.4), and then resuspended in staining buffer. Following this, cells were mixed with 5 µl annexin-V-FITC and 5 µl propidium iodide (PI). After 10 min, the mixtures were analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) and in-built software. Annexin V-positive and PI-negative cells were regarded as apoptotic cells.
4
Quantifying SH-SY5Y Cell Apoptosis via Annexin V-FITC
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An Annexin V-fluorescein isothiocyanate (FITC) cell apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.) was applied to detect SH-SY5Y cell apoptosis. In short, after transfection with indicated plasmids for 48 h, SH-SY5Y cells (10,000) were harvested and fixed by 4% formaldehyde at room temperature for 30 min. SH-SY5Y cells were then permeabilized using 0.2% Triton X-100 in PBS (Sigma-Aldrich; Merck KGaA) for 5 min at room temperature. Subsequently, cells were incubated with 5 µl Annexin V-FITC and 5 µl propidium iodide at room temperature for 10 min in the dark. Finally, the apoptosis rate of SH-SY5Y cells was analyzed by a BD FACSCalibur flow cytometry (BD Biosciences) using CellQuest software (version 3.0; BD Biosciences). Early apoptosis was assessed using the following formula: Cell apoptosis rate = number of cells in quadrant 3/number of cells in all quadrants.
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