Monocyte apoptosis was induced with 300 nM staurosporine (Calbiochem, Bad Soden, Germany) or 25 μM ABT-737 (Selleckchem, Houston, TX) and evaluated by measuring TMRE (Molecular Probes, Eugene, OR) MFI or the frequencies of Annexin V (Immunotools, Friesoythe, Germany) positive cells with flow cytometry using a FACS Calibur flow cytometer and CellQuest software (BD Biosciences, Heidelberg, Germany). In certain experiments, monocytes were pretreated with the inhibitors wortmannin (WM; PI3K inhibitor, Sigma, St. Louis, MO), LY294002 (PI3K inhibitor, Sigma), SH-6 (AKT inhibitor, Santa Cruz, Heidelberg, Germany) or U0126 (MEK/ERK inhibitor, Sigma) at indicated concentrations for 1 h. For immunofluorescence staining, polyclonal anti-cleaved caspase 3 antibody (5 μg/ml; rabbit IgG; Abcam) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:100; Invitrogen) were used. Nuclei were stained with TO-PRO-3 iodide (Molecular Probes). Confocal microscopic analysis was performed using a LSM510 META confocal laser scanning microscope and ZEN 2012 imaging software (Carl Zeiss MicroImaging, Jena, Germany).
Annexin 5
Manufactured by Immunotools
Sourced in Germany
Annexin V is a laboratory reagent used to detect and quantify apoptosis, a programmed cell death process. It binds to phosphatidylserine, a phospholipid that is exposed on the outer membrane of cells undergoing apoptosis. This binding allows for the identification and analysis of apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (2 mentions)
Abt 737, by Selleck Chemicals (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Annexin 5 binding buffer, by BD (2 mentions)
Annexinv binding buffer, by R&D Systems (2 mentions)
7 aad, by R&D Systems (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Ly294002, by Merck Group (1 mentions)
U0126, by Merck Group (1 mentions)
Lsm 510 meta confocal laser scanning microscope, by Zeiss (1 mentions)
Zen 2012 imaging software, by Zeiss (1 mentions)
Annexin 5 fitc, by Immunotools (1 mentions)
Macsquant, by Miltenyi Biotec (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ebioscience fixable viability dye efluor 450, by Thermo Fisher Scientific (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Propidum iodide pi, by Merck Group (1 mentions)
Tmre assay kit, by Abcam (1 mentions)
15 protocols using annexin 5
1
Monocyte Apoptosis Induction and Analysis
2
Cell Cycle and Cell Death Analysis
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Cell cycle and cell death analyses were performed 72 h after the treatment of cells with indicated doses as previously described [32 (link)]. Briefly, collected attached and floating cells were stained with a Nicoletti buffer (50 µg/µl propidium iodide (PI), 0.1% sodium citrate, and 0.1% Triton X-100) for cell cycle analysis. Numbers of dead cells were determined after incubation with annexin V-FITC (Immunotools, Friesoythe, Germany) in annexin V binding buffer and PI (2 μg/mL). Flow cytometry analysis was performed using a Miltenyi MACSQuant® analyzer (Milteny Biotec GmbH, Bergisch Gladbach, Germany) and MACSQuantify software.
3
FACS-Based Cell Death Mechanism Analysis
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For FACS-based analysis of cell death mechanisms, 1.5 × 105 A549 cells were transfected per 12-well. Supernatants of TRAIL-treated A549 cells were collected and recombined with the respective detached cells. These cells were washed with serum-free PBS and stained for 30 min in the dark with FITC-labelled Annexin V (31490013 × 2, 1:20, Immunotools, Friesoythe, Germany) and ebioscience Fixable Viability Dye eFluor 450 (1:2000, Invitrogen, Carlsbad, California, USA). Afterwards, cells were fixed for 10 min with 4% (w/v) paraformaldehyde (Sigma-Aldrich, Taufkirchen, Germany) diluted in PBS and then resuspended in 250 µL of Annexin V staining buffer (0.01 M HEPES, 0.14 M NaCl and 2.5 mM CaCl2, pH 7.5). Flow cytometric measurements were performed with a Gallios flow cytometer (Beckman Coulter, Indianapolis, Indiana, USA). Data were analyzed using the FlowJo software (v.10, BD Biosciences, Mississauga, ON, USA).
4
CFLAR Knockdown Impacts Cell Death
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CFLAR down-regulation was performed using commercially available siRNA assays according to manufacturer’s protocol (CFLAR MISSION® esiRNA, Sigma-Aldrich, Saint-Louis MO, USA), using RNAiMAX lipofectamine as a transfection reagent (Invitrogen, Carlsbad CA, USA). Cells were seeded in duplicates, grown to approximately 60% confluence, transfected with siRNA or scrambled control, grown for another 48 hours, serum deprived overnight and exposed to CSE for 4 hours. Subsequently CSE was washed away and replaced by CSE and serum free medium for 24 hours. The levels of the DAMPs dsDNA and RNA were measured in cell free supernatant using the Quant-iT™ Pico- and Ribo-Green® dsDNA Assay Kits respectively (Invitrogen). The percentage of viable, apoptotic and necrotic cells were determined using an Annexin-V (Immunotools, Friesoythe, Germany) and Propidum Iodide (PI; Sigma-Aldrich, Saint Louis, USA) staining for flow cytometry. Annexin-V/PI double negative cells were designated as viable cells, Annexin-V positive and PI negative cells were designated as apoptotic and all PI positive cells, either Annexin-V positive or negative, were designated as necrotic cells.
5
Annexin V Apoptosis Assay
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6
Mitochondrial Membrane Potential and Apoptosis
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In order to detect changes in the mitochondrial membrane potential (MMP), the tetramethylrhodamine ethyl ester (TMRE) assay kit (Abcam, Cambridge, UK) was used and the externalization of phosphatidylserine (PS) was determined using Annexin-V (Immunotools, Friesoyhte, Germany) staining. See the Online Supplementary Appendix for further information.
7
Apoptosis Kinetics of CD4+ T Cells
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CD4+ T cells were isolated by using MicroBeads (Miltenyi Biotech, catalogue number: 130-049-201) from spleens and LNs of 5-week-old Bcl-3TOE mice and control littermates. Triplicates of 1 × 105 CD4+ T cells were cultured for 4 days in T-cell media at 37 °C. Each day cells were counted, stained for AnnexinV (Immunotools, catalogue number: 31490016) and 7-aminoactinomycin D (BD Pharmingen, catalogue number: 559925) according to the manufacturer's instruction and analysed by FACS.
8
Apoptosis, Cell Cycle, and Erythroid Markers
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To detect apoptotic cells with exposed phosphatidylserine, cells were incubated for 30 min with FITC-conjugated Annexin-V (#31490013, Immunotools) and analyzed using FACSCalibur flow cytometer (BD Biosciences). For cell cycle analysis, cells were fixed in 70% cold ethanol, washed with PBS, treated with RNase A, and stained for 30 min with 40 μg/ml propidium iodide in the dark, at room temperature. DNA content was analyzed by measuring fluorescence intensity through flow cytometry. For analysis of membrane erythroid markers, cells were stained for 30 min with FITC conjugated anti-CD235a (Glycophorin A) antibody (#559943 BD Biosciences) and APC conjugated anti-CD71 (#551374 BD Biosciences). Fluorescent signals were measured using FACSCalibur.
9
Fluorescent Indole Cytotoxicity Assay
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680C91 ((E)-6-fluoro-3-[2-(3-pyridyl)vinyl]-1H-indole), ribonuclease-A, CHPx, anti β-tubulin monoclonal antibody, TRI Reagent were from Merck (KGaA, Darmstadt, Germany); Annexin V was from ImmunoTools GmbH (Gladiolenweg 2; Germany); mouse monoclonal anti-TDO antibody was from NovusBio (Bio-Techne SRL, MI, Italy); propidium iodide (PI) (BD Biosciences); high D-glucose DMEM, RPMI 1640, M199 and PBS were from Euroclone S.p.A. (MI, Italy).
10
Annexin V Apoptosis Assay in Ba/F3/gp130 Cells
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Ba/F3/gp130 cell lines were washed 3 times with PBS. For the 24 h analysis, 2.5 x 105 cells and for the analysis after 48 h, 1.25 x 105 cells were used per well and incubated with the indicated cytokines. For the condition ethanol + Hyper-IL-6 or GFP-Fc cells were only incubated with Hyper-IL-6 or GFP-Fc. Ethanol was added to the washing steps right before the measurement. After the indicated time points, cells were washed twice with ice-cold PBS and if indicated also with 70% ethanol. Cells were resuspended in 300 μL Annexin V binding buffer (BD Bioscience, Franklin Lakes, USA) and 0.5 μL Annexin V (ImmunoTools, Oldenburg, Germany) were added. Cells were vortexed and incubated at room temperature for 15 min in the dark. Then 1 μL 7-AAD (R&D Systems, Minneapolis, USA) was added and 400 μL of AnnexinV binding buffer. Analysis was carried out by flow cytometry (BD FACSCanto II flow cytometer, BD Biosciences, San Jose, USA). Data was evaluated using FlowJo_V10 (FlowJo LLC, Ashland, USA).
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