Allophycocyanin-conjugated annexin V and 7-aminoactinomycin D (BD Biosciences, San Jose, CA, USA) were used to analyze the cell apoptosis rate. Huh7-shNC and Huh7-shPYCR1 cells were washed twice with PBS and treated with 0.25% pancreatin (without EDTA) to facilitate digestion. After adjusting the cell density to 1 × 105/mL, the cells were incubated with fluorescent antibodies at room temperature for 25 min, then pelleted, and resuspended. The samples were analyzed using a flow cytometer (BD Biosciences) and the FlowJo software.
Allophycocyanin conjugated annexin 5
Manufactured by BD
Sourced in United States
Allophycocyanin-conjugated annexin V is a fluorescently labeled protein used for the detection and quantification of apoptotic cells. Annexin V binds to phosphatidylserine, which is exposed on the surface of cells undergoing apoptosis. The allophycocyanin label provides a fluorescent signal that can be detected using flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
7 aminoactinomycin d, by BD (2 mentions)
Annexin 5 binding buffer, by BD (2 mentions)
Hank balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Facsvantage se, by BD (1 mentions)
Pe conjugated anti human cd45, by BD (1 mentions)
Annexin 5, by Merck Group (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions)
Annexin 5, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Surface antibodies, by Thermo Fisher Scientific (1 mentions)
Pharm lyse lysing buffer, by BD (1 mentions)
7 amino actinomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
7 protocols using allophycocyanin conjugated annexin 5
1
Annexin V and 7-AAD Apoptosis Assay
2
Annexin V Binding Assay for RBC
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Preoperative arterial blood (fresh RBCs) and salvaged RBCs from 10 patients in the IBS group were centrifuged at 800g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended in PBS to a concentration of 5 × 106 cells/mL. Cells (5 × 106) were seeded into three 6-cm centrifuge tubes. The cells were plated and cultured at 37°C on a platform rotating at 80 rpm. To determine what fraction of these cells bound Annexin-V, cells were harvested after 0, 24, 48, or 72 hours of culture, and diluted to 1 × 106 cells/mL using Hank balanced salt solution (Invitrogen). To the resuspended cells (100.0 μL, 1 × 105 cells) was added 3.0 μL allophycocyanin-conjugated Annexin V (BD Bioscience), and the mixture was incubated at room temperature for 15 minutes in the dark. To this mixture was added 400.0 μL 1× binding buffer, and then flow cytometry of at least 50,000 events was performed.
3
Annexin V Apoptosis Assay
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Cells were seeded in six-well plates overnight. The next day, cells were treated with the indicated amount of drug(s) or vehicle control (DMSO). Incubation time was 15 hours for etoposide or 48 hours for DRA alone or DRA with sensitizers. To prepare samples for flow cytometry, each well of cells was washed twice with ice-cold PBS and resuspended in 1X annexin V binding buffer [10 mM Hepes, 140 mM NaCl, and 2.5 mM CaCl2 (pH 7.4); BD Biosciences]. Allophycocyanin-conjugated annexin V was used to measure surface exposure of phosphatidylserine, and 7-aminoactinomycin D was used as a viability probe (BD Biosciences). Cell samples were analyzed at 20,000 counts per sample using BD FACSVantage SE.
4
Quantifying Apoptosis in Primary AML Samples
5
Assessing CAR T Cell Cytotoxicity
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Cytotoxicity determined by Annexin V staining
To detect the cytotoxic effect of CAR T cells to target cells, we used Annexin V staining to analyze carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cell apoptosis by flow cytometry. Target cells were labeled with five µmol/l CFSE (BD Biosciences) in one mL PBS for 10 minutes at 37°C, then washed three times with culture medium containing 10% fetal bovine serum. ICC cells were cocultured with Mock or CAR T cells at an effector to target (E:T) ratio of 1:1. Following 18 hours of culture, cells were stained with allophycocyanin-conjugated Annexin V (BD Biosciences) and the percentage of dead target cells (defined as CFSE+ Annexin V+) was determined by a flow cytometric analysis (FACSAria II, BD Biosciences).
Cytotoxicity determined by cell counting kit-8 assay
6
Murine Peripheral Blood Flow Cytometry
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For murine peripheral blood flow cytometry analysis, peripheral blood was collected by retro-orbital bleeding and incubated with surface antibodies (eBioscience) in staining buffer (phosphate-buffered saline (PBS) supplemented with 0.5% FBS). Peripheral blood cell suspensions were treated with Pharm Lyse Lysing Buffer (BD Biosciences) and washed twice with PBS. For apoptosis assays, cells were incubated with allophycocyanin-conjugated Annexin V (BD Bioscience) for 15 min at RT in 1X Annexin V Binding Buffer (BD Bioscience) following staining with 7-aminoactinomycin (eBioscience). Data were acquired on a FACSCanto I and results were analyzed using FlowJo Version 10 (FlowJo). The list of antibodies used for flow cytometry analysis of mouse and human cells is indexed in Supplementary Data 4 .
7
Neutrophil Apoptosis and Macrophage Phagocytosis
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Human neutrophils were isolated from Ficoll-Hypaque pellets through dextran erythrocyte sedimentation and lysis of contaminating erythrocytes by incubation with ice-cold ammonium chloride (0.15 M) and potassium bicarbonate (0.01 M) solution. Neutrophils were resuspended at 1×106 cell/ml in 10% fetal bovine serum (FBS) - RPMI 1640 medium, labeled with 2.5 μM carboxyfluorescein succinimidyl ester (CFSE; Sigma-Aldrich), and incubated for 20 hours at 37°C in 5% CO2. Allophycocyanin-conjugated annexin V (BD Biosciences) and propidium iodide (PI; Sigma-Aldrich) were used to measure apoptosis by flow cytometry. The composition of neutrophils routinely obtained after incubation was 77.50 ± 10.05% for early apoptotic cells (ACs) (annexin V+ PI+), 4.59 ± 2.34% for late ACs (annexin V+ PI+), and 0.38 ± 0.29% for necrotic cells (annexin V+ PI+). Macrophages were differentiated in X-Vivo™15 medium supplemented with 10% human AB serum, 5% FBS, L-glutamine, penicillin and streptomycin, in the presence or absence of GW9662 (10 μM), for 6 days. In some experiments, cells were cultured in the presence of IL-4 (20 ng/ml) to obtain M2a macrophages. On day 6, apoptotic neutrophils were added for 30 minutes to cultured macrophages at a 5:1 ratio. Flow cytometry was used to quantify percentages of CD14-labeled macrophages that phagocytosed CFSE-labeled ACs.
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