Flow cytometry analysis of NOK cells was used to assay for cell viability and mechanism of cell death. NOKs were grown to semi confluence (1.5 x 104 cells per well) in 96-well plates (0.33 cm2 area) (Nunc, Rochester, NY), and treated with PrtFAC (10, 50, and 100 μM) for 48 h. Post treatment, adherent cells were trypsinized and washed with 1X PBS and centrifuged (at 500 g for 5 min) in sterile FACS 5 ml centrifuge tubes. The supernatant was discarded and the cells were stained for flow cytometry analysis using pacific blue™ Annexin-V and 7-Aminoactinomycin D (7-AAD) [Biolegend, San Diego, CA], according to the manufacturer’s instructions. Test volumes were decreased for staining in a 96 well plate. Briefly, cells were resuspended in 10 μl of Biolegend Annexin V binding buffer then stained with 0.5 μl of pacific blue™ Annexin V and 1 μl of 7-AAD for 15 min at room temperature in the dark. Cells were washed with 200 μl of Annexin-V binding buffer then resuspended in 1% paraformaldehyde. TRAIL protein (20 μM) was used as a positive control, while Z-VAD-FMK (20 μM), a pan-caspase inhibitor, was used as a caspase control. Data acquisition and analysis of apoptotic cells were performed using the MACSQuant 10 Analyzer (MiltenyiBiotec, Inc, Auburn CA) and FlowJo vX Data Analysis Software (FlowJo, Ashland, OR).
Annexin 5 pacific blue
Manufactured by BioLegend
Sourced in United States
Annexin V-Pacific Blue is a fluorescent dye-conjugated protein used for the detection of apoptotic cells. It binds to phosphatidylserine, which is exposed on the cell surface during the early stages of apoptosis. The Pacific Blue fluorescent dye is used to label the Annexin V protein, allowing for the visualization and quantification of apoptotic cells using flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 binding buffer, by BioLegend (6 mentions)
7 aad, by Thermo Fisher Scientific (5 mentions)
Macsquant vyb, by Miltenyi Biotec (5 mentions)
7 aad viability staining solution, by BioLegend (4 mentions)
Propidium iodide, by Miltenyi Biotec (4 mentions)
7 aad, by BioLegend (3 mentions)
Annexin 5 binding buffer, by BD (3 mentions)
Annexin 5 fitc, by BioLegend (3 mentions)
Accutase, by Thermo Fisher Scientific (3 mentions)
Annexin 5, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Annexin 5 alexa647, by BioLegend (2 mentions)
7 aad, by Merck Group (2 mentions)
Cd4 pe, by BD (2 mentions)
Celltrace violet ctv, by Thermo Fisher Scientific (2 mentions)
Cd4 apc, by BD (2 mentions)
Cd4 efluor 450, by BD (2 mentions)
Cell fixation permeabilization kit reagents, by BD (2 mentions)
Lsrii platform, by BD (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
46 protocols using annexin 5 pacific blue
1
Analyzing NOK Cell Viability and Apoptosis
2
Evaluating Apoptosis in Hematopoietic Stem Cells
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Cell apoptosis was evaluated using Pacific Blue Annexin V (Biolegend, 640918) and 7-AAD Viability Staining Solution (BioLegend, 420403). In detail, GE-HSPCs were stained with the following antibodies mix for 15 min at 4°C: CD34 PE (Miltenyi Biotec 130-081-00), CD90 APC (BD Biosciences, 559869), and CD133/1 PECy7 (Miltenyi Biotec, 130-101-652). Cells were then washed with diluted 1:10 10X Annexin V Binding Buffer (BD Pharmingen, 556454) upon surface marker staining and stained with Pacific Blue Annexin V (Biolegend, 640918) and 7-AAD Viability Staining Solution (BioLegend, 420403) for 15 min at RT in the dark. After staining, cells were washed in 1X Annexin V Binding Buffer and acquired in 10 min. All samples were run on BD FACSCanto II cytometer (BD Biosciences). At least 10,000 were recorded. Analysis of flow cytometry results was performed using FlowJo software.
3
Annexin V and 7-AAD Apoptosis Assay
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Collected cells were incubated in annexin V binding buffer (BioLegend, 422201) with Pacific Blue annexin V (BioLegend, 640917) and 7-AAD (Thermo Fisher Scientific, 00699350) at 4 °C for 15 min. Cells were measured using a MACSQuant VYB (Miltenyi Biotec) using MACSQuantify software 2.13.0 and analyzed using FlowJo v10.7.1 (BD). The gating strategy can be found in Supplementary Figure 1 .
4
Annexin V-PI Apoptosis Assay
5
Leukemia Cell Apoptosis Quantification
6
Cell Viability and Apoptosis Assessment
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Cell viability was measured using the Cell Counting Kit 8 (APExBIO, K1018) according to the manufacturer's instructions. Annexin V/propidium iodide (PI) staining was used to determine apoptosis. Pacific Blue Annexin V (640918) was purchased from Biolegend. PI (ST511) was purchased from Beyotime. Annexin V/PI staining was performed according to the manufacturer's instructions. The fluorescence was measured using Becton Dickinson LSRFortessa X20 flow cytometry and analyzed with FlowJo Version 10.6 software.
7
Flow Cytometry Apoptosis Assay
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After 24 h of incubations with the stimuli, cells were collected. Briefly, cells were washed with PBS and then detached with accutase (Gibco, Life Technologies) and resuspended in 100 μL of 1× annexin V-binding buffer. The reagent Pacific-Blue Annexin V (5 μL, BioLegend, San Diego, CA, USA) was added to the cell suspension for 15 min and incubated at room temperature. Then 400 μL of 1× annexin V-binding buffer was added to each sample. Propidium iodide (5 μL, Miltenyi Biotec, Bergisch, Germany) was added to the suspension before the cytometry analysis. Ten thousand cell per sample were collected in a MACSQuant-VYB (Miltenyi Biotec, Surrey, UK). Data analysis was carried out by using the FlowJo software.
8
Apoptosis Quantification of EpCAM+/CD133+ Cells
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The cells were trypsinized and stained with APC/Fire 750 EpCAM antibody (1:20 dilution), VioBright FITC CD133/1 antibody (1:50 dilution), Pacific Blue Annexin V (Biolegend, #640918, 1:20 dilution), and 7-AAD (Biolegend, #420404, 1:20 dilution) for 15 min at room temperature in the dark for the determination of cell apoptosis. The apoptotic cells were quantified using flow cytometry. The corresponding isotype-matched control antibodies were used to control for background fluorescence.
9
Annexin V Apoptosis Assay
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Cells were collected after incubation for 24 h with the stimuli. Cells were washed with PBS, then detached with Accutase (Gibco, Life Technologies), and resuspended in 100 μl of 1X Annexin-binding buffer. Pacific-Blue Annexin V (5 μl, BioLegend) was added to the cell suspension and after a 15-min incubation at room temperature, 400 μl of 1X Annexin-binding buffer was added. Before analysis by flow cytometry, propidium iodide (5 μl, Miltenyi Biotec) was added to the suspension. 10,000 events per sample were collected in a MACSQuant-VYB (Miltenyi Biotec, Surrey, UK). Data analysis was performed using the FlowJo software.
10
CAR-NK-92 Cell Cytotoxicity Assay
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Cytotoxicity was analyzed by performing 7-amino-actinomycin D (7-AAD) and Annexin V assay. Target OCI-Ly7 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) using a Cell Trace Cell Proliferation kit (Thermo Fisher Scientific, Inc.), as previously described (31 ). CAR-NK-92 cells were co-cultured with CFSE-labeled target cells at an effector-to-target ratio of 2:1. After incubation at 37°C overnight, the cells were washed with Annexin V binding buffer (BD Biosciences) and incubated at 37°C for 30 min. CAR-NK-92 cells were cultured at 37°C for 4 h, pre-treated with human FcR blocking reagent at 1:200 dilution (Miltenyi Biotec GmbH) for 5 min, and stained with 7-AAD Viability Staining Solution (BioLegend, Inc.) and Pacific Blue™ Annexin V (BioLegend, Inc.) for 15 min on ice. The CFSE+ target cells were evaluated for early and late apoptosis using flow cytometry using CytoFLEX (Beckman Coulter, Inc.). The data were analyzed using FlowJo v10 software (Tree Star, Inc.).
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