HuCCt-1 cells were seeded in 60 mm dishes, washed after 24 h once with serum-free media and incubated with 2.0, 1.25, or 0.6 µM napabucasin for additional 24 h. Untreated control samples were included in each experimental series. The Annexin V assay was performed following the manufacturer’s protocol (Biolegend, San Diego, CA, USA), and analyzed by flow cytometry (Cell Lab Quanta SC, Beckman Coulter, Brea, CA, USA) using the Kaluza Analysis 1.3 software (Beckman Coulter).
Annexin 5 assay
Manufactured by BioLegend
Sourced in United States
The Annexin V assay is a laboratory tool used to detect and quantify cellular apoptosis or programmed cell death. Annexin V is a protein that binds to phosphatidylserine, a molecule that is externalized on the surface of cells undergoing apoptosis. This assay allows researchers to measure the level of apoptosis in cell samples.
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Lab products found in correlation
Mts assay, by Promega (2 mentions)
Facscanto 2, by BD (2 mentions)
Modfit software, by Verity Software House (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Cell lab quanta sc, by Beckman Coulter (1 mentions)
Kaluza analysis 1, by Beckman Coulter (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Mouse t cell isolation kit, by STEMCELL (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Triton x, by Merck Group (1 mentions)
Np 40, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Prism 7, by GraphPad (1 mentions)
Bovine serum albumin (bsa), by BD (1 mentions)
Mzfliii stereomicroscope, by Leica (1 mentions)
Facs canto 2, by Verity Software House (1 mentions)
5 protocols using annexin 5 assay
1
Napabucasin-Induced Apoptosis in HuCCt-1 Cells
2
Evaluating diABZI Effects on TAMCs
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In vitro generated TAMCs (1×106 cells/well) in 6-well plate were treated with diABZI at 200 nM. After 6 h of treatment, cells were washed and collected for RNA isolation using the RNeasy Plus Mini Kit (Qiagen). Total RNA was quantified by Nanodrop (Thermo Scientific), and cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). Gene expression was analyzed by quantitative PCR analysis (Bio-Rad). The primer sequences are listed in Supplementary Table 1 . Alternatively, flow cytometric analysis was performed 24 h after treatment. To determine the cytotoxicity of diABZI to T cells, T cells were isolated using mouse T cell isolation kit (STEMCELL Technologies) and seeded at 2×105 cells/well in 96-well U bottom plates with the addition of Dynabeads mouse T cell activator CD3/CD28 (Fisher) and IL-2 (PeproTech) at 50 U/ml. Cells were treated with diABZI at 200 nM for 48 h and collected for annexin V assay (BioLegend) using flow cytometry.
3
Apoptosis and Cell Cycle Analysis in AML
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To measure apoptosis, Annexin V assay (BioLegend) was performed as described previously19 (link). To assess the cell cycle status, AML cells were stained with DAPI (5 μg/mL) and 0.1% NP40 (Sigma). Live cells were enumerated by staining with propidium iodide (20 μg/mL) (Sigma). Data were analysed using FlowJo 10.0.8 (Tree Star, Inc) software and graphed using GraphPad Prism 7 (GraphPad Software Inc, CA). For intracellular flow cytometry, AML cells were fixed with 1% PFA (Thermofisher) for 12′ on ice. Cells were permeabilised with 2% BSA (Thermofisher) and 0.1% Triton-X (Sigma) for 20′ on ice, washed, and stained for 30′ on ice in the dark with anti-human GATA2-PE (IC2046P) (R&D systems). Cells were then washed twice with 2% BSA PBS and analysed in a BD LSR Fortessa IV (BD).
4
Evaluating Cell Proliferation, Apoptosis, and Cell Cycle
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Cell proliferation/viability was evaluated by the tetrazolium dye (MTS) assay (Promega, Madison, WI). Each cell line was plated at a seeding density to give logarithmic growth over the course of the assay in a 96-well tissue culture plate. The concentration of final product is measured by absorbance at 490 nm and is proportional to the viable cell number in each well. For soft agar assays, 5 × 103 to 104 cells were suspended in 0.8% low melting point agarose (Difco Laboratories Inc., Detroit, MI) at room temperature, mixed with an equal volume of 2x concentrated culture media, and plated onto an agarose bed consisting of 2% low melting point agarose and the same medium. After 3–6 weeks, colonies were stained with neutral red and were enumerated using a Leica MZ FLIII Stereomicroscope (Leica, Germany).
Apoptosis was determined using the Annexin V assay (Biolegend, San Diego, CA) and cell cycle was analyzed using the Propidium Iodide assay (Invitrogen, MA). After treatment, cells were collected and stained with Annexin V-FITC and/or propidium iodide solution as per the manufacturer’s protocol and analyzed by flow cytometry. Cell death was recorded in a FACSCanto II (BD Biosciences, Mississauga, ON) in the total population (10,000 cells) and data were analyzed using FACSCanto II software and ModFit Software (Verity Software House, Topsham, ME).
Apoptosis was determined using the Annexin V assay (Biolegend, San Diego, CA) and cell cycle was analyzed using the Propidium Iodide assay (Invitrogen, MA). After treatment, cells were collected and stained with Annexin V-FITC and/or propidium iodide solution as per the manufacturer’s protocol and analyzed by flow cytometry. Cell death was recorded in a FACSCanto II (BD Biosciences, Mississauga, ON) in the total population (10,000 cells) and data were analyzed using FACSCanto II software and ModFit Software (Verity Software House, Topsham, ME).
5
Evaluating Cell Proliferation and Apoptosis
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Cell proliferation/viability was evaluated by the tetrazolium dye (MTS) assay (Promega, Madison, WI). Each cell line was plated at a seeding density to give logarithmic growth over the course of the assay in a 96-well tissue culture plate. The concentration of final product was measured by absorbance at 490 nm, which is proportional to the viable cell number in each well. Apoptosis was determined using the Annexin V assay (Biolegend, San Diego, CA), and cell cycle was analyzed using the propidium iodide assay (Invitrogen, Waltham, MA). Cells were seeded in culture plates 24 hours before incubation with RG7388 for the indicated doses and times. Cells were collected and stained with Annexin V-FITC and/or propidium iodide solution as per the manufacturer's protocol and analyzed by flow cytometry. Cell death was recorded in a FACSCanto II (BD Biosciences) in the total population (10,000 cells), and data were analyzed using FACSCanto II software and using ModFit Software (Verity Software House, Topsham, ME).
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