Levels of apoptotic and dead cells were determined by flow cytometry using eBioscience™ Annexin V Apoptosis Detection Kit APC (Thermo Fisher, Darmstadt, Germany). The differentiation between late-apoptotic or necrotic and early-apoptotic cells was facilitated by staining the cells with the fluorescent dye propidium iodide and allophycocyanin (APC)-coupled Annexin V prior to flow cytometric analysis [70 ]. For this purpose, HCT116 IDH1+/+ or IDHR132H/+ cells were seeded and treated with VC (1 mM) or ML309 (10 µM) alone and with combination of both for 48 h. Cells grown in unamended medium were used as vehicle control, and cells treated with TNFα and cycloheximide (20 ng/ml, 25 µg/ml, respectively) 24 h prior to cell harvest were used as positive control for the induction of apoptosis. The protein synthesis inhibitor cycloheximide was included in the positive control to induce pro-apoptotic effects of TNFα. Per run 10,000 events were counted and analysed on a FACSCanto II (BD Biosciences, Heidelberg, Germany). FlowJo software (Treestar, Ashland, USA) was used for data analysis.
Ebioscience annexin 5 apoptosis detection kit apc
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The EBioscience™ Annexin V Apoptosis Detection Kit APC is a laboratory instrument designed to detect and measure apoptosis, a type of programmed cell death. The kit utilizes Annexin V, a calcium-dependent phospholipid-binding protein, which has a high affinity for phosphatidylserine (PS), a molecule that is translocated from the inner to the outer leaflet of the plasma membrane during apoptosis. The APC (Allophycocyanin) fluorescent dye is conjugated to the Annexin V protein, allowing for the detection of apoptotic cells using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Facscanto 2, by BD (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Fortessa x 50, by BD (2 mentions)
Accuri c6 cytometer, by BD (2 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Control igg, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti c3b antibody, by Thermo Fisher Scientific (1 mentions)
Incyte 3, by Merck Group (1 mentions)
Guava easycyte flow cytometer, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Cytexpert software, by Beckman Coulter (1 mentions)
Facscalibur, by BD (1 mentions)
Ebioscience fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
Trypsin edta 0.05, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
Lsr 2, by BD (1 mentions)
Facsdiva 8, by BD (1 mentions)
29 protocols using ebioscience annexin 5 apoptosis detection kit apc
1
Apoptosis Quantification by Flow Cytometry
2
Apoptosis Assay of Lapatinib and Ketoconazole
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Cells were treated with lapatinib and/or ketoconazole for 48 hours in monolayer cultures. They were then stained for Annexin V and PI using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) or the eBioscience™ Annexin V Apoptosis Detection Kit APC (Thermo Fisher Scientific) according to the manufacturer's protocol.
3
Viral Infection Induces Apoptosis
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Viable B-cells (control) from non-infected cultures and infected GFP+ cells were sorted by FACS at 24 hpi and 5x105 cells were seeded per P24-wells in B-cell medium without chCD40L for 24 h. At 48 hpi, cells were washed once in phosphate-buffered saline (PBS) and incubated with the Fixable Viability Dye eFluor 780. Cells were then washed twice in PBS 1X, BSA 0.1%. Apoptotic cells were stained using the eBioscience Annexin V Apoptosis Detection Kit APC according to the manufacturer’s protocol (Thermo Fisher Scientific). Cell viability and apoptosis analyses were performed by cytometry. Early apoptotic cells were defined as Annexin V-positive / eFluor-negative and late apoptotic cells were defined as Annexin V-positive/eFluor-positive.
4
Quantification of Apoptosis by Flow Cytometry
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Levels of apoptotic and dead cells were determined by flow cytometry using eBioscience™ Annexin V Apoptosis Detection Kit APC (Thermo Fisher, Darmstadt, Germany). Briefly, 2 x 105 HCT116 cells/well were seeded into 6-well plates (TPP, Trasadingen, Switzerland). After 24 h, the cells were incubated with the substances at the indicated concentrations for 72 h. Incubation with TNFα (20 ng/mL) and cycloheximide (20 μg/mL) for 72 h served as positive control for apoptosis induction. Subsequently, the cells were washed and stained with Annexin V antibody and propidium iodide (PI) according to the manufacturer’s instructions. The cells were distinguished between viable cells (Annexin V− / PI−), early apoptotic cells (Annexin V+ / PI−), late apoptotic/necrotic cells (Annexin V+ / PI+) and late necrotic cells (Annexin V− / PI+). Per run 10,000 events were counted and analyzed on a FACSCanto II (BD Biosciences, Heidelberg, Germany). FlowJo software (Treestar, Ashland, USA) was used for data analysis.
5
C3b Detection and Apoptosis Analysis
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For the detection of C3b, cells were treated with culture media containing 10% pooled human serum before trypsinization. The cells were incubated with mouse monoclonal anti-C3b antibody (Thermo Scientific, Rockford, IL) or control IgG (Santa Cruz, Santa Cruz, CA) for 30 min on ice. After washing, allophycocyanin (APC)-conjugated goat anti-mouse IgG (R&D systems, Minneapolis, MN) was added for 30 min at 4 °C. After washes, cells suspended in 1% FBS/PBS were analyzed using a Guava easyCyte Flow Cytometer and the InCyte 3.1 software (Merck Millipore, Bedford, MA). For apoptosis analysis, eBioscience Annexin V apoptosis detection kit APC (Thermo Scientific) was used as manufacturer’s instructions.
6
Assessing Non-Canonical NF-κB Pathway
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The human CLL cell line MEC1, the splenic marginal zone lymphoma cell lines SSK41 and VL51, the mantle cell lymphoma cell lines MAVER-1, Z-138 and JEKO-1, the human HEK-293T cell line, as well as primary CLL cells were used in functional experiments. The entire non-canonical NF-κB pathway was assessed by western blot analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to analyze the non-canonical NF-κB signature. Primary CLL were exposed to fludarabine and venetoclax for 24-48 h and apoptosis was measured using the eBioscience Annexin V Apoptosis Detection Kit APC (ThermoFisher). Details are supplied in the Online Supplementary Methods.
7
Quantifying Apoptosis Levels in Transduced Cells
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A total of 2x105 cells/ml were cultured in a 6-well plate after 3 days of transduction. The cell fusion degree was detected to be ~85% on the 5th day following transduction. The cells were trypsinized and centrifuged at 4˚C for 5 min at 300 x g, washed twice with D-Hanks solution (pH=7.2-7.4) and once with the 1X Binding Buffer, then resuspended in 1X Binding Buffer at a density of 1x106 cell/ml. Next, the 100 µl of the cell suspension were incubated at 25˚C for 15 min in the dark with the 5 µl of eBioscience™ Annexin V Apoptosis Detection Kit APC (Thermo Fisher Scientific, Inc.) Finally, the samples were analyzed in a flow cytometer using Guava easyCyte HT Version 8 system (EMD Millipore).
8
Apoptosis and Cell Cycle Analysis
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A total of 5x105 cells per well were seeded in 6-well plates and cultured overnight. The cells were treated with drugs for 24 hrs. Apoptosis and cell cycle were investigated using the eBioscience™ Annexin V Apoptosis Detection Kit APC according to the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, USA) by FACS Calibur flow cytometer (BD, Franklin Lakes, USA). The FACS data were analyzed using FLOWJO v10.1.
9
Annexin V-Based Apoptosis Assay
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Cells were plated at a concentration of 500.00 cells/ml and treated as indicated. After incubation for 24 h, apoptotic cells were measured by flow cytometry using Ebioscience™ Annexin V Apoptosis Detection Kit APC according to the kit instruction (Cat. 88-8007-74, Thermo Fisher Scientific). Cells positive for annexin-V were counted as apoptotic cells.
10
Apoptosis Assay of Lung Cancer Cells
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The eBioscience™ Annexin-V Apoptosis Detection Kit (APC) (Thermo Fisher Scientific) was used to measure apoptosis in lung cancer cells treated with AICAR. Briefly, the cells (H1975, PC9, H23, and A549) were plated in a 24-well plate and treated with AICAR (1 mM) for 0, 4, 7, and 16 h (H1975) or 7 h (PC9, H23, and A549). Then the cells were collected and incubated with Annexin-V conjugated with APC (1:40) in 200 µL binding buffer for 12 min at room temperature. Afterward, cells were stained with 7AAD. The fluorescence-labelled cells were measured with a CytoFLEX flow cytometer (Beckman Coulter) using APC (Ex/Em:633/660) and PI channels (546/647). 10,000 independent events were analysed by CytExpert software (Beckman Coulter).
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