Cells with an lncRNA BANCR overexpression rate of >200% and their controls were subjected to the apoptosis assay. In brief, cell suspensions (6×104 cells/ml) were prepared using serum-free medium. Cells were cultivated in a 6-well plate with 10 ml cell suspension in each well, followed by the addition of 5, 10, 20 or 40 mM d-glucose. After the cells were cultivated for 48 h (15 (link)), digestion with 0.25% trypsin was performed, and the cells were collected and suspended in DMEM medium. subsequently, Annexin V-fluorescein isothiocyanate (FITC; Dojindo, Kumamoto, Japan) and propidium iodide (PI) staining was performed and apoptotic cells were detected by flow cytometry using WOLF Cell Sorter (NanoCellect Biomedical, Inc., San Diego, CA, USA).
Annexin 5 fluorescein isothiocyanate fitc
Manufactured by Dojindo Laboratories
Sourced in Japan
Annexin V-fluorescein isothiocyanate (FITC) is a protein-fluorophore conjugate used for the detection and quantification of apoptosis. Annexin V has a high affinity for phosphatidylserine, a phospholipid that is externalized during the early stages of apoptosis. The FITC fluorescent label allows for the visualization and analysis of apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Dojindo Laboratories (2 mentions)
Apoptotic cell detection kit, by Roche (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Cellquest software, by BD (1 mentions)
Facscan, by BD (1 mentions)
Facsuite software version 10, by BD (1 mentions)
6 well plate, by Corning (1 mentions)
Facscalibur, by BD (1 mentions)
Fcs express 6 flow cytometry software, by De Novo Software (1 mentions)
Trypsin, by Sangon (1 mentions)
7 protocols using annexin 5 fluorescein isothiocyanate fitc
1
Apoptosis Assay of BANCR-Overexpressing Cells
2
Apatinib Induces Apoptosis in Cells
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The cells were seeded at 5000 cells per well in 96-well plates and incubated overnight. After a particular treatment, the cell viability was determined using Cell Counting Kit-8 (Dojindo, Japan), according to the manufacturer’s instructions. The cell survival rates are expressed as the means ± SD from three independent experiments.
Apoptosis was examined by flow cytometric analysis. The cells were treated with certain concentrations of apatinib for the indicated durations. Both floating and adherent cells were collected, stained with Annexin V–fluorescein isothiocyanate (FITC), and propidium iodide (Dojindo, Kumamoto, Japan), and further analyzed with a flow cytometer (FACScan, BD Biosciences, San Jose, CA, USA) equipped with Cell Quest software (BD Biosciences). Apoptosis was also determined using the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) apoptotic cell detection kit (Roche, Basel, Switzerland), according to the manufacturer’s instructions. Apoptosis was expressed as the mean ± SD from three independent experiments.
Apoptosis was examined by flow cytometric analysis. The cells were treated with certain concentrations of apatinib for the indicated durations. Both floating and adherent cells were collected, stained with Annexin V–fluorescein isothiocyanate (FITC), and propidium iodide (Dojindo, Kumamoto, Japan), and further analyzed with a flow cytometer (FACScan, BD Biosciences, San Jose, CA, USA) equipped with Cell Quest software (BD Biosciences). Apoptosis was also determined using the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) apoptotic cell detection kit (Roche, Basel, Switzerland), according to the manufacturer’s instructions. Apoptosis was expressed as the mean ± SD from three independent experiments.
3
Apoptosis Measurement by Flow Cytometry
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We selected cells in logarithmic phase, prepared cell suspensions, and placed them in a 5% CO2, 37 °C incubator for 48 h. Cells were trypsinized and centrifuged. Cell suspensions were incubated with annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis kit (DOJINDO, Japan). After incubation, 400 μL 1× annexin V binding buffer was added into the cell suspensions and then analyzed using a flow cytometer (BD Bioscience, USA). The excitation wavelength was 488 nm and the emission wavelength was 530 nm. Approximately 10,000 cells were counted for each measurement.
4
Apoptosis Assessment of Cultured Cells
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Cells were suspended in serum-free medium (6×104 cells/ml) and were transferred to a 6-well plate (10 ml cell suspension/well). After cell culture for 48 h, cells were subjected to 0.25% trypsin digestion. Subsequently, cells were stained with Annexin V-fluorescein isothiocyanate (FITC; 1:50; Dojindo Molecular Technologies, Inc.) and propidium iodide (PI, 1:50). Flow cytometry was performed to detect apoptotic cells. Data were analyzed using BD FACSuite™ software version 1.0 (BD Biosciences, San Jose, CA, USA). Cell apoptotic rates were calculated based on the sum of % Annexin V-FITC+/PI- and % Annexin V-FITC+/PI+ cells.
5
Evaluating Trichostatin A Effects on Cultured Cells
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Cultured cells (786-O, ACHN, and A498) used to evaluate proliferation were grown on 96-well plates at a concentration of 3×103 cells per well, whereas cells for apoptotic evaluation were grown on 6-well plates (Corning Incorporated) at a concentration of 1.2×106 cells per well. Subsequently, cells were incubated in an atmosphere of 5% CO2 and 95% air for 24 h prior to renewing the media containing 0.5, 1.0, or 2.0 µmol/l TSA, as appropriate. Cells treated with medium without TSA were used as a negative control, whereas medium without the cells was used as a blank control. The cell medium was renewed every 24 h for all groups. The proliferation of cells was evaluated at 0, 24, 48, and 72 h using CCK-8, as described above. Apoptosis was examined at 48 h using Annexin V-fluorescein isothiocyanate (FITC) (Dojindo Molecular Technologies, Inc.,) and flow cytometry (Instrument: Bio-Rad Laboratories, Inc; Software: NoveExpress™), in accordance with the manufacturer’s protocol.
6
Cell Cycle and Apoptosis Analysis
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2 × 105 H3122CR cells were treated with siRNA after growing in 6-well plates for 24 h. To analyze the cell cycle phase, cells treated for 42 h were stained with propidium iodide (PI) (Dojindo, Japan). To evaluate cell apoptosis, Annexin V–fluorescein isothiocyanate (FITC) and PI were used to stain the cells after treatment for 48 h (Dojindo, Japan). Flow cytometry was used to analyze prepared samples (BD FACSCalibur).
7
Cardiomyocyte Apoptosis Assay with NKILA
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lncRNA NKILA expression reached 200% and knockdown rate reached 50% at 24 h after the transfection of siRNA and vectors. Following 24 h transfection, suspensions of primary human cardiomyocyte cells at a density of 5×104 cells/ml were prepared using serum-free cell culture medium (as aforementioned) supplemented with 20 mM D-glucose, before being subsequently seeded into 6-well plates at 2 ml/well. The cells were then cultured for 48 h, followed by digestion using 0.25% trypsin (Sangon Biotech Co., Ltd.) before being subjected to Annexin V-Fluorescein isothiocyanate (FITC; Dojindo Molecular Technologies, Inc.) and propidium iodide (PI; Dojindo Molecular Technologies, Inc.) staining. Apoptotic cells were detected using a flow cytometer. Data were analysed using FCS Express 6 Flow Cytometry Software (De Novo Software).
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