Cell apoptosis was evaluated using Annexin V/PI apoptosis detection kit (Abcam) using the instruction book and examined by flow cytometry. Briefly, cells were seeded on the six-well plates and incubated with DSG for 24 h, then trypsinized, and resuspended using the binding buffer. Subsequently, 5 μL of Annexin V-FITC and 5 μL of PI were added and incubated for 15 min. Apoptotic cells were analyzed by an FC500 flow cytometer.
Annexin 5 pi apoptosis detection kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The Annexin V/PI apoptosis detection kit is a laboratory product used to detect and quantify apoptosis in cell populations. It contains Annexin V, a protein that binds to phosphatidylserine, and propidium iodide, a DNA-binding dye. This kit allows for the identification of cells in different stages of apoptosis through flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Wst 1 reagent, by Roche (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Fc receptor blocking agent, by BioLegend (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Revertaid m mulv reverse transcriptase, by Takara Bio (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
9 protocols using annexin 5 pi apoptosis detection kit
1
Annexin V-FITC/PI Apoptosis Assay
2
Annexin V-FITC and Propidium Iodide Apoptosis Assay
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Cells in each treatment group were supplemented with serum-free DMEM medium to induce apoptosis. Briefly, cells were digested with 0.25% trypsin at 37˚C for 2 min. to prepare a single cell suspension and washed twice with ice cold PBS. Cells were incubated with Annexin V-FITC (cat. no. ab14085; Abcam) for 10 min at room temperature (25˚C). Following this, the probe solution, 50 µg/ml propidium iodide (PI; cat. no. ab14083; Abcam) was added directly to the cell suspension. Cells were incubated for 30 min at room temperature in the dark prior to flow cytometry analysis. Subsequently, the cells were washed with fresh serum-free DMEM. The fluorescent intensity was detected via flow cytometry (ZE5 Cell Analyzer; cat. no. 12004279; Bio-Rad Laboratories, Inc.) and apoptosis rate was measured a by Annexin V-PI Apoptosis Detection kit (Abcam), according to the manufacturer's protocol. Data were analyzed using FlowJo software (version 10.0; FlowJo LLC).
3
Cell Viability Assay Protocol
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Dulbecco’s modified Eagle’s medium (DMEM) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, USA). The Annexin V/PI apoptosis detection kit was purchased from Abcam (Cambridge, UK), and WST-1 reagent was purchased from Roche (Mannheim, Germany). All other chemicals used in this study were purchased from Sigma (St. Louis, USA) unless otherwise stated.
4
Flow Cytometry Analysis of CD14+ PBMC Apoptosis
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CD14+ PBMC apoptosis was analysed via the flow cytometry method (FCM), using an Annexin V-PI Apoptosis Detection Kit (Abcam). Briefly, cells were collected six days after the induction of differentiation, washed with phosphate-buffered saline (PBS), and suspended in 500 µl of binding buffer. They were then incubated (37°C, 10 min) with Annexin V, stained with propidium iodide (PI), and analysed via the FCM to determine their relative quantitative rate of apoptosis.
5
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, SNK-6 cells were cultured in 0.01 mmol/l doxorubicin, or not. After 24 h, the cells were stained in 50 mg/ml propidium iodide, 0.05% Triton x-100, 0.1 mg/ ml RNase A, and 1x PBS at 37˚C for 30 min in the dark. The stained cells were suspended in 500 ml PBS for flow cytometric analysis.
Cell apoptosis was analyzed using an Annexin V-PI apoptosis detection kit (Abcam, Cambridge, UK). Briefly, transfected cells were washed with PBS and resuspended in 500 µl of binding buffer containing Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). After incubation on ice for 10 min, cells were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). The relative number of apoptotic cells was calculated.
Cell apoptosis was analyzed using an Annexin V-PI apoptosis detection kit (Abcam, Cambridge, UK). Briefly, transfected cells were washed with PBS and resuspended in 500 µl of binding buffer containing Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). After incubation on ice for 10 min, cells were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). The relative number of apoptotic cells was calculated.
6
Annexin V-PI Apoptosis Assay in NSCLC
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Apoptosis of NSCLC cells was detected by the Annexin V-PI Apoptosis Detection Kit (BioVision, California, USA). Briefly, cells were harvested by trypsinization and collected in centrifuge tubes, and washed with ice-cold PBS. Then, the cells were suspended in 500 ml of binding buffer and incubated with Annexin V-FITC and propidium iodide (PI). The apoptosis rate was analyzed by flow cytometry (FACSCalibur; BD, Franklin Lakes, NJ, USA).
7
Multiparametric Flow Cytometry of Immune Cells
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Cells were trypsinized, resuspended in PBS supplemented with 2% BSA, incubated with Fc receptor blocking agent (Biolegend, 101302), and stained with fluorescently labeled antibodies for 30 min on ice: FITC-conjugated CD8α, CD4, and PE-conjugated NK1.1, CD45, and PerCP/Cyanine5.5-conjugated CD3, Gr-1, and BV421-conjugated F4/80 and BV510-conjugated CD11b, and APC-conjugated CD107a, CD54, and PE/CY7-conjugated PD-1, PD-L1. For intracellular detection, cells were stimulated with PMA and ionomycin (BD Biosciences) for 6 h, fixed and permeabilized with Cytofix/Cytoperm Kit (BD Biosciences) and stained with specific antibodies: BV510-conjugated IFNγ. For apoptosis assay, an annexinV/PI apoptosis detection kit was used according to the manufacturer’s protocol (Biovision). The data were analyzed with FlowJo software (version 10.5; Tree Star).
8
Apoptosis Detection and Gene Expression
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RPMI 1640 culture medium, FBS, PBS, penicillin, streptomycin, and Trypsin/EDTA solution were all purchased from Gibco (Rockville, MD, USA). MTT, Trypan blue, and dimethyl sulfoxide (DMSO) were procured from Sigma Aldrich (St. Louis, MO, USA). The Annexin V/PI apoptosis detection kit was obtained from BioVision (San Francisco, CA, USA). The RevertAid M-MuLV Reverse Transcriptase and the cGMP Direct Immunoassay kit were obtained from Takara Bio Inc. (Dalian, China) and R&D Systems (Minneapolis, MN, USA), respectively. All other materials were of analytical grade.
9
Apoptosis and Cell Viability Assays
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The culture media, Roswell Park Memorial Institute medium (RPMI 1640), trypan blue, EDTA, trypsin, penicillin, streptomycin, phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were all purchased from Gibco (Rockville, MD, USA). The Annexin V/PI Apoptosis Detection Kit was obtained from BioVision (San Francisco, CA, USA). The cGMP Direct Immunoassay Kit was procured from R&D Systems (Minneapolis, MN, USA). The 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The RevertAid M-MuLV Reverse Transcriptase (RT) was procured from Fermentas (Vilnius, Lithuania). All other materials were of analytical grade and were obtained locally.
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