At the indicated incubation time, floating cells were collected in culture supernatants and adherent cells were harvested by trypsinization. After collection, cells were washed twice with cold 1× PBS, and about 2 × 105 cells were resuspended in 100 μl binding buffer (FITC Annexin V Apoptosis Detection Kit, Biolegend). Subsequently, 5 μl FITC Annexin V (FITC Annexin V Apoptosis Detection Kit, Biolegend) and 10 μl propidium iodide was added to the cell suspension. Cells were gently vortexed and incubated in the dark for 15 min at RT. Thereafter, another 400 μl Annexin V binding buffer was added to each tube. Cells were analyzed using a FACS CALIBUR (BD Sciences) flow cytometer. Dot plots were generated using the FlowJo software.
Fitc annexin 5 apoptosis detection kit
Manufactured by BioLegend
Sourced in United States
The FITC Annexin V Apoptosis Detection Kit is a laboratory product that utilizes fluorescein isothiocyanate (FITC)-labeled Annexin V to detect apoptotic cells. Annexin V binds to phosphatidylserine, a phospholipid that translocates to the outer cell membrane during the early stages of apoptosis. The kit provides a simple and efficient method for identifying and quantifying apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (13 mentions)
Facscan, by BD (8 mentions)
7 aad, by BioLegend (6 mentions)
Facscanto 2, by BD (5 mentions)
Propidium iodide, by Merck Group (4 mentions)
Cellquest software, by BD (4 mentions)
Bradford reagent, by Bio-Rad (4 mentions)
Facscanto 2 flow cytometer, by BD (3 mentions)
Cell staining buffer, by BioLegend (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Fc500 system flow cytometer, by Beckman Coulter (2 mentions)
Cyto ex ow cytometer, by Beckman Coulter (2 mentions)
Facsaria 2, by BD (2 mentions)
Guava easycyte flow cytometer, by Merck Group (2 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (2 mentions)
Propidium iodide solution, by BioLegend (2 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Facsuite system, by BD (2 mentions)
Facsverse cytometer, by BD (2 mentions)
118 protocols using fitc annexin 5 apoptosis detection kit
1
Annexin V-FITC Apoptosis Assay
2
Cellular Viability and Apoptosis Assay
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To analyze cellular viability, apoptotic and dead cells were quantified using the FITC Annexin V apoptosis detection kit and 7-AAD (Biolegend), following the manufacturer’s instructions. BEAS-2B, HK-2, Huh-7, Caco-2, and HUVEC cells were plated in 6-well plates at a seeding density of 3 × 106 cells/well and a range of concentrations of VPA (1, 2, 4, 8, and 16 mM) were added for 24 h. After that, cells were removed and stained using the FITC-Annexin V Apoptosis detection kit with 7AAD (Biolegend), following the manufacturer’s instructions, and analyzed on a Gallios Flow Cytometer (Beckman Coulter, Inc.). Early apoptotic cells were considered as Annexin V + 7AAD- and late apoptotic/necrotic cells as Annexin V + 7AAD + .
The effect of VPA on cell proliferation was analyzed with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Huh-7 and HK-2 cells were seeded at a density of 5 × 103 cells/well in 96-well plates at the same VPA concentrations, as described above, for 24 h. Depending on the experimental requirements, medium was then replaced, and cells were cultured for a further 24 or 48 h or kept unreplaced for direct analysis. During the final 4 h, 0.5 mg/ml of MTT substrate was added to the cell culture. After that, the medium was removed, and formazan crystals were dissolved with DMSO. Absorbance at 570 nm was read by a spectrophotometer (Bio-Rad).
The effect of VPA on cell proliferation was analyzed with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Huh-7 and HK-2 cells were seeded at a density of 5 × 103 cells/well in 96-well plates at the same VPA concentrations, as described above, for 24 h. Depending on the experimental requirements, medium was then replaced, and cells were cultured for a further 24 or 48 h or kept unreplaced for direct analysis. During the final 4 h, 0.5 mg/ml of MTT substrate was added to the cell culture. After that, the medium was removed, and formazan crystals were dissolved with DMSO. Absorbance at 570 nm was read by a spectrophotometer (Bio-Rad).
3
Apoptosis Assessment by Flow Cytometry
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At the indicated incubation time, floating cells were collected in culture supernatants and adherent cells were harvested by trypsinization. After collection, cells were washed twice with cold 1X PBS, and about 2x10 5 cells were resuspended in 100 μl binding buffer (FITC Annexin V Apoptosis Detection Kit, Biolegend). Subsequently, 5 μl FITC Annexin V (FITC Annexin V Apoptosis Detection Kit, Biolegend) and 10 μl propidium iodide was added to the cell suspension. Cells were gently vortexed and incubated in the dark for 15 min at RT. Thereafter, another 400 μl Annexin V binding buffer was added to each tube. Cells were analysed using a FACS CALIBUR (BD Sciences) flow cytometer. Dot plots were generated using the FlowJo software.
4
Apoptosis assay of COS, GA, and COS-GA in SW620 cells
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SW620 cells were counted and seeded in a 6-well plate at 2 × 105 cells/well in 2 mL of the completed medium/well before incubation at 37 °C in a CO2 incubator for 24 h. The cells were subsequently treated with COS, GA and COS–GA at varying concentrations (62.5, 122, and 250 µg/mL) for 24 and 48 h. Apoptotic cells were then quantified by staining the cells with the FITC Annexin V apoptosis detection kit with propidium iodide (PI; Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, cells were harvested, washed twice with cell-staining buffer (0.5% bovine serum albumin in phosphate-buffered saline), and resuspended in Annexin V Binding Buffer. Thereafter, 100 µL of the cell suspension was mixed with 5 µL of FITC Annexin V dye and 10 µL of PI solution before incubating at 25 °C for 15 min. The volume of the cell suspension was then adjusted by adding 400 µL of Annexin V Binding Buffer, and the sample was subjected to flow cytometry analysis using FACSAria II (BD Biosciences, San Jose, CA, USA).
5
Cell Viability Assay for ccRCC and Normal Cortex
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Cell viability of metabolically treated ccRCC and normal cortex primary cultures was assessed. 2.5 × 105 cells were seeded in 6-well plates and treated with 2DG or Etomoxir for 72 hours. Cell viability was evaluated with FITC Annexin V Apoptosis detection kit and Propidium Iodide (PI) (Biolegend, San Diego, CA) according to the manufacturer’s instructions. Briefly, after two washes with cold PBS, the cells were resuspended in 100 µl of Annexin V Binding Buffer. The cell solution was then incubated with 5 µl of FITC Annexin V and 10 µl of PI for 15 minutes at room temperature in the dark. After the incubation, 200 µl of Annexin V Binding Buffer were added. FACS analysis was performed on 10000 events with MoFlo Astrios Cell Sorter and Kaluza software (Beckman Coulter srl, Milano, Italy). Viable (Annexin V and PI negative), dead (PI positive) and apoptotic (Annexin V positive) cells in treated and untreated samples were expressed as percentage with respect to total number of analysed events.
6
Annexin V-PI Apoptosis Detection Assay
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Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidyl serine (PS) in a calcium-dependent manner. In viable and healthy cells, PS is located in the inner membrane leaflet, but during early apoptosis, it is translocated to the external leaflet and becomes available for annexin V binding. Annexin V binding alone cannot differentiate between apoptotic and necrotic cells. The addition of PI enabled viable, early apoptotic, late apoptotic, and necrotic cells to be distinguished. All the operations were performed according to the manufacturer’s instructions (FITC Annexin V Apoptosis Detection kit, BioLegend, USA). Briefly, the cells in 6 designed groups were rinsed with PBS and resuspended in a binding buffer [4 (link)]. Then 5 mL of Annexin V (20 mg.mL-1) and 10 mL of propidium iodide (PI, 50 mg.mL-1) were added to the suspension and incubated for 15 min at room temperature and analyzed by flow cytometer.
7
Apoptosis Analysis by Flow Cytometry
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Apoptosis was analyzed by cytometric analysis, using FITC Annexin V Apoptosis Detection Kit (Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, cells were trypsinized, washed twice with cold PBS, and 106 cell/mL were resuspended in 1X binding annexin V-FITC (0.25 µg/mL) and propidium iodide (PI) (1 µg/mL) were added to cell suspension and incubated, protected from light, for 10 min at room temperature. Samples were analyzed using Guava easyCyteflow cytometer (Merck Millipore, Darmstadt, Germany). For each sample, 5000 events were acquired [44 (link)]. Annexin V-FITC is detected as a green fluorescence and propidium iodide is detected as a red fluorescence. Early apoptosis is defined by annexin V+/PI− staining, late apoptosis is defined by annexin V+/PI+ staining, and necrosis is defined by annexin V−/PI+ staining).
8
Annexin V-FITC Apoptosis Assay
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The detection of apoptosis was assessed using FITC Annexin V Apoptosis Detection Kit (Biolegend, California, USA), as previously described [27 (link)]. T24 and 5637 cells after different treatment were resuspended in Annexin V binding buffer at a concentration of 1.0 × 107 cells/mL. Subsequently, 5 μL Annexin V-FITC and 5 μL PI solution were added to the cell resuspension and the cells were incubated for 30 mins in the dark. Stained cells were centrifuged and washed twice with Annexin V binding buffer and resuspended in 400 μL Annexin V binding buffer. The percentage of apoptotic events was analyzed by BD FACS CantoTM II Flow Cytometer (BD Biosciences).
9
Erythroid Differentiation Inhibitors Protocol
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Three inhibitors were used in this study. LDN27291 was purchased from TOCRIS. GK921 was from MedChemExpress and Z-DON was from Zedira. The inhibitors were added respectively to in vitro cultured mouse fetal liver derived erythroid cells at the beginning (D0) and day 2 of differentiation (D2).
The following antibodies were from BD Bioscience: CD16/CD32 (mouse BD Fc Block), FITC Rat Anti-mouse Ter119, PE Rat Anti-mouse CD44, APC-Cy7 Rat Anti-mouse GR1, APC-Cy7 Rat Anti-mouse CD45, APC-Cy7 Rat Anti-mouse CD11b and APC Rat Anti-mouse CD71. Rabbit Anti-mouse TGM2 and PE Anti-mouse TGM2 were from Abcam and Novusbio respectively. FITC Annexin V Apoptosis Detection Kit were from Biolegend.
The following antibodies were from BD Bioscience: CD16/CD32 (mouse BD Fc Block), FITC Rat Anti-mouse Ter119, PE Rat Anti-mouse CD44, APC-Cy7 Rat Anti-mouse GR1, APC-Cy7 Rat Anti-mouse CD45, APC-Cy7 Rat Anti-mouse CD11b and APC Rat Anti-mouse CD71. Rabbit Anti-mouse TGM2 and PE Anti-mouse TGM2 were from Abcam and Novusbio respectively. FITC Annexin V Apoptosis Detection Kit were from Biolegend.
10
Annexin V Apoptosis Assay for Melanoma
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Flow cytometry was performed to evaluate cell early apoptosis using the FITC Annexin V Apoptosis Detection kit (BioLegend, Inc.), according to the manufacturer's protocol. Melanoma cells cultured in 6-well plates were rinsed three times using cell staining buffer, resuspended in Annexin V Binding Buffer (5×106 cells/ml) and incubated using 5 µl FITC Annexin V and 7-AAD Viability Staining Solution for 15 min at room temperature in the dark. Finally, the cells were mixed with Annexin V Binding Buffer and analyzed with a FACScan flow cytometry system (BD Biosciences) and FlowJo software (version 10; FlowJo LLC). Flow cytometry analysis experiments were repeated ≥3 times.
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