Viability analysis was done using APC-conjugated Annexin V (Biolegend Cat. No. 640920 and propidium iodide (Miltenyi Biotec Cat. No. 130–093-233). Briefly, adhered RH30 cells following trypsinization are transferred into a U-bottomed 96-well plate for staining. Cells are stained for 20 min at room temperature with APC-conjugated Annexin V (Biolegend cat # 640,920; 1:100 dilution) and propidium iodide (Miltenyi Biotec cat# 130–093-233; 1:100 dilution) diluted in Annexin V Binding Buffer (Cat. No. 422201) followed by 2X washes in warm Annexin V Binding Buffer and analysis by flow cytometry.
Apc conjugated annexin 5
Manufactured by BioLegend
Sourced in United States
APC-conjugated Annexin V is a fluorescently-labeled protein that binds to phosphatidylserine, a phospholipid expressed on the surface of apoptotic cells. This product can be used in flow cytometry applications to detect and quantify apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 binding buffer, by BioLegend (8 mentions)
Facscanto 2, by BD (3 mentions)
Facsverse cytometer, by BD (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
7 aad, by Merck Group (2 mentions)
Annexin 5 binding buffer, by Miltenyi Biotec (2 mentions)
Propidium iodide, by Miltenyi Biotec (2 mentions)
Propidium iodide, by BioLegend (2 mentions)
Fitc conjugated anti cd4 antibody, by BioLegend (1 mentions)
Cell staining buffer, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
M csf, by Cell Signaling Technology (1 mentions)
2 7 dichlorofluorescin diacetate dcfda cellular ros detection assay kit, by Abcam (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Fcs express software, by De Novo Software (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
31 protocols using apc conjugated annexin 5
1
Apoptosis and Necrosis Analysis
2
Apoptosis and Necrosis Analysis
3
Apoptosis Detection in T Cells
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PBMCs were harvested for detection of apoptosis of CD4+ and CD8+ cells using flow cytometry. Briefly, after blocking, cells were incubated on ice with FITC-conjugated anti-CD4 antibody (Biolegend, San Diego, CA, USA) and PE-conjugated anti-CD8 antibody (Biolegend, San Diego, CA, USA) for 30 min in the dark. The cells were washed and incubated with APC-conjugated annexin V (Biolegend, San Diego, CA, USA) for 15 min in the dark and were analyzed.
4
Detecting Apoptosis in Tumor Cells
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The apoptosis detection assays were performed using APC-conjugated annexin V and propidium iodide (PI) solution (both, BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, the GiNKs were incubated with control IgG, anti-KIR2DL1, or anti-KIR2DL2/3 antibody for 30 min prior to use. Then, the U87MG and T98G cells were exposed to the GiNKs at E:T ratios of 1:1 in the presence or absence of 1 µg/mL control IgG, anti-KIR2DL1, or anti-KIR2DL2/3 for 24 h. Following incubation, the floating cells and detached cells were collected and washed twice by cold Cell Staining Buffer (BioLegend) and resuspended in Annexin V Binding Buffer (BioLegend) at a concentration of 106 cells/mL. Then, 5 μL APC-conjugated annexin V and 10 μL PI solution were added to the cell suspension of 100 µL Binding Buffer. The cells were gently mixed and incubated for 15 min at room temperature (25 °C) in the dark. Finally, 400 μL Binding Buffer was added and the apoptotic tumor cells were detected with a BD FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using FlowJo version 10 (BD Biosciences).
5
Apoptosis Assay for Cell Viability
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Cells were incubated for 72 hours with various concentrations of each compound, and then stained with APC-conjugated Annexin V (BioLegend) and propidium iodide (Sigma-Aldrich). Apoptotic cells, defined as APC-conjugated Annexin V-positive cells, were analyzed using a FACSVerse cytometer (BD Biosciences). Data are expressed as the mean ± SD of three independent experiments and were analyzed using FlowJo software (Tree Star).
6
Fatty Acid Modulation of Macrophage ROS
7
Evaluating NTR-Mediated Cell Death
8
Apoptosis Analysis of LCMV-Specific CD8+ T Cells
9
Mammosphere Assay and Apoptosis Analysis
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To evaluate apoptotic cell death, tumor cells were stained with a combination of propidium iodide and APC-conjugated Annexin V (10 μg/ml, BioLegend), and analyzed by flow cytometry at the core facility. For the tumorsphere assay, MCF-10A cells were allowed to grow in suspension (24–well ultra-low adhesion plate) in a stem cell culture medium, which consisted of mammary epithelial basal medium (MEBM) (Lonza, Basel, Switzerland) supplemented with B27 mixture (Life Technologies, Carlsbad, CA), 20 ng/ml bFGF, 20 ng/ml EGF and 4 µg/ml Heparin, over a span of 5–7 days, and followed by imagining microscopically. The size, number and morphologies of mammosphere or colonies were quantified by analyzing the photographed representative fields using Nikon NIS-Elements Advanced Analysis Software [31] (link).
To generate stable cell lines from primary tumors in the MMTV-Wnt1 mice, fresh tumor tissues were digested in collagenase overnight. After filtering with 40 µm strainers, cell mixtures were cultured for 3–5 generations in a F12/DMEM medium supplemented with 5% fetal bovine serum, 10 ng/ml EGF, 0.25 µg/ml hydrocortisone, 100 ng/ml Cholera toxin and 10 µg/ml insulin.
To generate stable cell lines from primary tumors in the MMTV-Wnt1 mice, fresh tumor tissues were digested in collagenase overnight. After filtering with 40 µm strainers, cell mixtures were cultured for 3–5 generations in a F12/DMEM medium supplemented with 5% fetal bovine serum, 10 ng/ml EGF, 0.25 µg/ml hydrocortisone, 100 ng/ml Cholera toxin and 10 µg/ml insulin.
10
Cytotoxicity Assay for NK Cells
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Sorted CD56dim and CD56neg NK cells (Figure S1 ) were resuspended in RPMI‐1640 medium containing 10% FCS with 100 IU/mL IL‐2 (Peprotech) and added to the targets at effector/target ratios from 10:1 to 0.6:1, each in duplicate. Cells were incubated for 4 hours at 37°C in a 5% CO2 incubator. Cells were stained with APC‐conjugated Annexin V (BioLegend). Percentages of Annexin V+ EGFP‐positive K562 cells were measured. Spontaneous lysis was determined by incubating target cells without effectors. Specific lysis was calculated as [(lysis in the well containing target cells and effector cells − spontaneous lysis)/ (100 − spontaneous lysis)] × 100. NK cells were incubated for 1 hour in the presence of 1 μg/mL nivolumab prior to coculture with PD‐L1‐K562 cells. Specific lysis was calculated as described above. Samples were acquired by flow cytometry (FACSCalibur; BD Biosciences), and the data were analyzed with FlowJo software ver. 7 (Tree Star).
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