An Annexin-V FITC-propidium iodide (PI) Apoptosis kit (Invitrogen: Thermo Fisher Scientific, Inc.) was used to determine the level of apoptosis. HepG2 cells were transfected with si-Igf2as and cultured for 48 h prior to the assay. These cells were collected and washed with PBS three times. The cells were resuspended in ice-cold 95% ethanol with 0.5% Tween 20. The fixed cells were washed in 1% BSA-PBS solution. Then, the cells were resuspended in 1X Annexin-V binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2]. Annexin-V FITC and PI were added to the cells at room temperature for 15 min in the darkness. Following incubation, the cells were filtered and analyzed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) within 1 h of staining. The number of cells in each quadrant was expressed as percentages of total stained cells. Data from a minimum of three independent repeats was used to quantify the number of apoptotic cells and graphed with GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA).
Annexin 5 fitc propidium iodide apoptosis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Annexin-V FITC-propidium iodide (PI) Apoptosis kit is a laboratory tool used to detect and quantify apoptosis, a programmed cell death process. The kit contains Annexin-V, a protein that binds to phosphatidylserine, and propidium iodide, a DNA-binding dye. This combination allows for the simultaneous identification of early apoptotic, late apoptotic, and necrotic cells through flow cytometry analysis.
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Lab products found in correlation
Cellquest pro software, by BD (2 mentions)
Prism 7, by GraphPad (1 mentions)
Facs flow cytometer, by BD (1 mentions)
Trypsin without edta, by Thermo Fisher Scientific (1 mentions)
Facscalibur system, by BD (1 mentions)
Modfit software version 4, by Verity Software House (1 mentions)
Lsrfortessa, by BD (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Cellquest pro software version 5, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest pro, by BD (1 mentions)
8 protocols using annexin 5 fitc propidium iodide apoptosis kit
1
Quantification of Apoptosis in HepG2 Cells
2
Annexin V-FITC/PI Apoptosis Assay
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Cell apoptosis was detected using the Annexin V-FITC/propidium iodide (PI) apoptosis kit (Invitrogen). Briefly, chondrocytes were cultured for 48 h, digested using trypsin without EDTA (Thermo Fisher Scientific), and washed with PBS. The cells were collected and adjusted to a cell density of 1 × 106 cells/ml. Then, cells were stained with Annexin V-FITC and PI for 15 min. At last, a BD FACS flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect the fluorescence intensity.
3
Flow Cytometric Analysis of Cardiomyocyte Apoptosis
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Primary cardiomyocytes were cultured in six-well plates at a density of 106 cells/well for 24 h and infected with Ad-miR-29a-3pi or Ad-NC (density, 107 viral genome particles) for 48 h at 37°C. Then, the cells were collected and washed with PBS three times (5 min/time). To determine cell apoptosis, an Annexin V-FITC-propidium iodide (PI) Apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used. In brief, the cells were washed with 1X Annexin V Binding Buffer (140 mM NaCl, 2.5 mM CaCl2 and 10 mM HEPES/NaOH, pH 7.4) at a concentration of 2–3×106 cells/ml. Then, the Annexin V-FITC and PI buffer was added and incubated with the cells at room temperature for 15 min. After treatment, the cells were filtered using a 300-mesh filter and analyzed by a BD FACSCalibur system (BD Biosciences) within 1 h of staining. Data were analyzed using ModFit software version 4.1 (Verity Software House, Inc.).
4
Apoptosis Assay of TA1 and AT1 Peptides
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The apoptosis assay was done in LNCaP and CWR cells following 20 μM treatment with peptide TA1 or AT1 for 4 days using annexin V-FITC/propidium iodide apoptosis kit (Invitrogen) or caspase 3/5 apoptosis kit (Invitrogen) following the manufacturer’s protocol. Cell apoptosis was analyzed by LSRFortessa (BD Biosciences) using appropriate controls.
5
Quantifying Apoptosis with Annexin V-FITC/PI
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An Annexin V-FITC/propidium iodide apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used for cell apoptosis analysis. Cells (5.0x105/ml) were seeded on a 75 mm plate for 24 h. After cells were treated with the reagents or H/R, medium was collected in a centrifuge tube and the cells were digested with trypsin (Gibco; Thermo Fisher Scientific, Inc.). Medium was mixed with the cell suspension, and the suspension was centrifuged using a Cence centrifuge (Changsha Xiangyi Centrifuge Instrument Co., Ltd.) at 1,200 x g for 5 min at ˚C. The cell pellet was then resuspended in PBS. Apoptosis kit reagents were added to the cells and the fluorescence was detected using a BD FACSCalibur flow cytometer (BD Biosciences) and BD CellQuest™ Pro Software version 5.1 (BD Biosciences). Procedures were conducted following the manufacturer's instructions.
6
Apoptosis Induction by Supernatant or 5-FU
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Three mL of growth medium including 1.2 × 105 cells was cultured in 6-well culture plates and incubated at growth condition. After 24 h, the cells were treated with 3 mL of the sterile growth medium containing determined dried materials of supernatant or 5-FU, as the positive control group, and incubated in the growth condition based on the determined time point. The treated/untreated control cells were detached by trypsin-EDTA, and supernatants were discarded by centrifugation at 900 rpm for 10 min. Finally, for detection of apoptosis, the cells were stained with Annexin V-FITC/Propidium iodide (PI) apoptosis kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions, and data analysis was conducted using CELL Quest Pro software (BD Biosciences, San Jose, CA, USA). After performing the flow cytometry, the cell populations were defined using quadrant gates. The number of cells in each quadrant represented quadrant 1 (Q1): necrotic cells (Annexin V−/PI+); quadrant 2 (Q2): late apoptotic cells (Annexin V+/PI+); quadrant 3 (Q3): early apoptotic cells (Annexin V+/PI−); and quadrant 4 (Q4): live cells (Annexin V−/PI−) [26 , 27 (link)]. Each experiment was repeated 2 times with triplicate samples. All of the analyses were performed using 150,000 cells at a rate of 900 cell/sec.
7
Apoptosis Assay of Cells
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Three mL of growth medium including 1.2 × 10 5 cells was cultured in 6-well culture plates and incubated at growth condition. After 24 h, the cells were treated with 3 ml of the sterile growth medium containing determined dried materials of supernatant or 5-FU, as the positive control group, and incubated in the growth condition based on the determined time point. The treated/untreated control cells were detached by trypsin-EDTA, and supernatants were discarded by centrifugation at 900 rpm for 10 minutes. Finally, for detection of apoptosis, the cells were stained with Annexin V-FITC/propidium iodide (PI) apoptosis kit (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions, and data analysis was conducted using CELL Quest Pro software (BD Biosciences, San Jose, CA, USA). Each experiment was repeated 2 times with triplicate samples. All of the analyses were performed using 150000 cells at a rate of 900 cell/sec.
8
Quantifying Apoptosis in Cell Lines
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Three mL of growth medium including 1.2×10 5 cells was cultured in 6-well culture plates and incubated at growth condition. After 24h, the cells were treated with 3 ml of the sterile growth medium containing determined dried materials of supernatant or 5-FU, as the positive control group, and incubated in the growth condition based on the determined time point. The treated/untreated control cells were detached by trypsin-EDTA, and supernatants were discarded by centrifugation at 900 rpm for 10 minutes. Finally, for detection of apoptosis, the cells were stained with Annexin V-FITC/Propidium iodide (PI) apoptosis kit (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions, and data analysis was conducted using CELL Quest Pro software (BD Biosciences, San Jose, CA, USA). After performing the ow cytometry, the cell populations were de ned using quadrant gates. The number of cells in each quadrant represented quadrant 1 (Q1): necrotic cells (Annexin V-/PI+); quadrant 2 (Q2): late apoptotic cells (Annexin V+/PI+); quadrant 3 (Q3): early apoptotic cells (Annexin V+/PI-); and quadrant 4 (Q4): live cells (Annexin V-/PI-) (26, 27). Each experiment was repeated 2 times with triplicate samples. All of the analyses were performed using 150000 cells at a rate of 900 cell/sec.
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