Annexin V-fluorescein isothiocyanate (FITC) kits (BD Biosciences, San Jose, CA, USA) were used to detect cell apoptosis in strict compliance with the instructions. Briefly, hPDLSCs were detached with 0.25% trypsin and centrifuged at 800 g and the supernatant was discarded. Then, the cells were washed with 0.01 mol/L PBS and resuspended in the binding buffer. Subsequently, 1 × 106 cells were stained with 5 μL Annexin V-FITC and 10 μL propidium iodide under conditions devoid of light. Flow cytometer was used to detect cell apoptosis.
Annexin 5 fluorescein isothiocyanate fitc kit
Manufactured by BD
Sourced in United States
The Annexin-V-fluorescein isothiocyanate (FITC) kit is a laboratory tool used for the detection and quantification of apoptosis, a programmed cell death process. The kit contains Annexin-V, a protein that binds to phosphatidylserine, a molecule that is exposed on the surface of cells undergoing apoptosis. The Annexin-V is conjugated with the fluorescent dye FITC, allowing for the visualization and analysis of apoptotic cells using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Flow cytometric analysis, by Beckman Coulter (1 mentions)
Novocyte, by Agilent Technologies (1 mentions)
Poly adp ribose polymerase parp, by Cell Signaling Technology (1 mentions)
Transwell invasion chamber, by Corning (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Crystal violet staining solution, by Beyotime (1 mentions)
Matrigel invasion chamber, by BD (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Ab19898, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Dxflex, by Beckman Coulter (1 mentions)
Facsaria 3 flow cytometry system, by BD (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
N cadherin, by Santa Cruz Biotechnology (1 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
21 protocols using annexin 5 fluorescein isothiocyanate fitc kit
1
Annexin V-FITC Apoptosis Assay
2
Annexin V-FITC Apoptosis Assay
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Annexin V/fluorescein isothiocyanate (FITC) kits (BD Biosciences) were utilized as per the manufacturer’s protocols. Following OGD treatment and transfection, PC12 cells were cultured in a 6-well plate. The cells were incubated with 100 μL binding buffer and 5 μL FITC-labeled Annexin V (20 μg/mL) in darkness for 15 min at ambient temperature. Then, 5 μL propidium iodide (PI; 50 μg/mL) was added for a 5-min incubation in the dark. Thereafter, a 400-μL binding buffer was added and immediately loaded onto a FACScan for flow cytometric quantitative detection (within 1 h).
3
Apoptosis and Lymphocyte Subset Analysis
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An annexin-V-fluorescein isothiocyanate (FITC) kit (BD Biosciences, San Jose, CA, United States) was used to measure the proportion of cells undergoing apoptosis. BRL 3A cells were treated with AT-1, collected, resuspended in 500 μl augmented binding buffer, and incubated for 15 min at 25°C in the dark in a reagent mix comprising 5 μl of the annexin-V-FITC conjugate and 10 μl of PI. The samples were analyzed using a flow cytometer (Novocyte, Agilent, United States) to determine the cell percentage at various phases.
For detecting lymphocyte subsets, 100 μl of the blood sample was stained with 10 μl of FITC-conjugated CD3, PE-conjugated CD4, and APC-conjugated CD8 reagent (Beckman Coulter), followed by incubation for 15 min in the dark for lysis of red blood cells. After washing, the cells were resuspended and subjected to flow cytometric analysis (Beckman). Lymphocytes were defined with their forward and side scatter characteristics. T lymphocytes were identified (CD3+) and then subdivided into CD4+ or CD8+ populations.
For detecting lymphocyte subsets, 100 μl of the blood sample was stained with 10 μl of FITC-conjugated CD3, PE-conjugated CD4, and APC-conjugated CD8 reagent (Beckman Coulter), followed by incubation for 15 min in the dark for lysis of red blood cells. After washing, the cells were resuspended and subjected to flow cytometric analysis (Beckman). Lymphocytes were defined with their forward and side scatter characteristics. T lymphocytes were identified (CD3+) and then subdivided into CD4+ or CD8+ populations.
4
Annexin-V/FITC Apoptosis Assay
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Apoptotic cells in two groups were identified by flow cytometry and the Annexin-V/Fluorescein Isothiocyanate (FITC) kit (BD Biosciences). A549 cells were collected and re-suspended, followed by incubation in 200 μl of binding buffer containing 5 μl of Annexin-V/FITC at room temperature in the dark for 10 minutes. After being centrifuged at 1,000g for 5 minutes at room temperature, the supernatant was discarded and the cells were re-suspended in 200 μl of fresh binding buffer. Then, 10 μl of PI was gently added and incubated on ice in the dark for two minutes. Finally, 400 μl of PBS was added and flow cytometric analyses were performed.
5
Apoptosis Assessment in Oxidative Stress
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To assess apoptosis, cells were initially cultured in MEM supplemented with 1 mM H2O2 for 1 hour to simulate oxidative stress conditions relevant to diabetic environments. Following this incubation, the medium was discarded and the cells were exposed to MEM containing various treatments. After 2 days, these cells were harvested, washed, and analyzed using an Annexin V/fluorescein isothiocyanate (FITC) kit from BD Biosciences. After staining cells with FITC Annexin V for 15 min while protected from light, they were stained with Propidium Iodide Staining Solution. The apoptosis levels were evaluated through flow cytometry using an Agilent Technologies instrument.
6
Apoptosis and Cell Cycle Analysis
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Results of the apoptosis assay were observed via flow cytometry performed using an Annexin V-fluorescein isothiocyanate (FITC) kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Cells were harvested, washed and resuspended with binding buffer. Cells were subsequently incubated with Annexin V-FITC then washed with cold PBS and resuspended. Finally, propidium iodide (PI) was added to mark apoptotic cells, which were then analyzed using a flow cytometer.
Cell cycle staging was also analyzed using flow cytometry. The cell suspension was fixed with 70% cold ethanol overnight at 4°C, then washed with cold PBS three times and stained with PI solution for 30 min in the dark at 37°C prior to being analyzed with a flow cytometer, to estimate the frequency of cells in the sub-G0/G1 phase. Each measurement was repeated in triplicate.
Cell cycle staging was also analyzed using flow cytometry. The cell suspension was fixed with 70% cold ethanol overnight at 4°C, then washed with cold PBS three times and stained with PI solution for 30 min in the dark at 37°C prior to being analyzed with a flow cytometer, to estimate the frequency of cells in the sub-G0/G1 phase. Each measurement was repeated in triplicate.
7
Evaluating miR-145's Effects on NSCLC
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The human NSCLC cell line A549 was purchased from the American Type Culture Collection (Manassas, VA, USA). RPMI-1640 medium, fetal bovine serum (FBS), PBS, bovine serum albumin (BSA), dimethylsulfoxide and Cell Counting Kit-8 (CCK-8) were purchased from Beijing Transgen Biotech Co., Ltd. (Beijing, China). Antibodies against matrix metalloproteinase (MMP)-2, MMP-9, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, poly (ADP-ribose) polymerase (PARP), β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Lipofectamine® 2000 and Opti-MEM were purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). AnnexinV/fluorescein isothiocyanate (FITC) kit and Matrigel invasion chambers were purchased from BD Biosciences (San Jose, CA, USA). The Transwell invasion chambers were purchased from Costar (Corning Life Sciences, Cambridge, MA, USA). Crystal violet staining solution was purchased from Beyotime Institute of Biotechnology (Haimen, China). Scrambled sequence and miR-145 mimic were purchased from Biotend (Shanghai, China).
8
Annexin V/PI Apoptosis Assay
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The number of apoptotic cells in the different treatment groups was examined using Flow cytometry analysis. The cells were stained with Annexin V/fluorescein isothiocyanate (FITC) kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Briefly, MA-C cells were cultured in 6-well plates, following OGD/RX treatment and transfection. The cells were collected,100 μl Binding Buffer and 5 μl fluorescein isothiocyanate (FITC)-labeled Annexin V (20 μg/ml) were added and incubated in the dark at room temperature for 15 min. Then, 5 μl propidium iodide (PI; 50 μg/ml) was added and incubated in the dark for 5 min. Thereafter, 400 μl of Binding Buffer was added and immediately subjected to FACScan for quantitative detection by flow cytometry (within 1 h).
9
Annexin-V-FITC Apoptosis Assay
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A PI kit and Annexin-V-Fluorescein isothiocyanate (FITC) kit (BD Biosciences, San Jose, CA, USA) were used following the manufacturer’s instructions. Pre-chilled PBS was used to wash the cells (2–3 × 105) twice, which were then suspended in binding buffer (100 µL) and 5 μL of FITC-conjugated annexin-V, and incubated in dark conditions at room temperature for 30 min. Thereafter, PI (100 μL) was added and incubation continued for 5 min, before the addition of Binding Buffer (400 µL). Subsequently, a flow cytometer CANTO™ II (BD Biosciences) was used to examine the cells and the data were examined with the aid of the FlowJo software (Becton Dickinson, Franklin Lakes, NJ, USA).
10
Apoptosis Analysis of Transfected RB Cells
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Flow cytometric analysis was performed on RB cells following transfection. About 2 × 105 cells were plated in 24 wells plate, transfected with 50 pm of antagomirs to all the three miRNAs (hsa-miR–106b-25 cluster). Flow cytometric analyses were performed after 48 hours of transfection, using the Annexin V-fluorescein isothiocyanate (FITC) Kit for apoptosis analysis according to the manufacturer’s protocol (BD Biosciences, Gurgaon, India).
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