For proliferation measurements, CFSE or CTe450 labeled cells were stained with Zombie aqua fixable viability dye (Biolegend) for 15 minutes, with surface antibodies for 20 min, then washed and fixed in 1% paraformaldehyde before flow cytometric analysis. For apoptosis assessment of PBMC and T cells, after surface staining, cells were stained with Annexin V (R&D Systems) per manufacturer’s instructions and were run immediately without fixation or were fixed in 1% PFA diluted in Annexin V binding buffer, containing CaCl2 to preserve Annexin V binding. To measure intracellular cytokines, cells were treated with GolgiPlug™ (BD BioScience) for the last 5 hours of culture, then stained with fixable viability dye and surface antibodies as above. Cells were then permeabilized using Invitrogen eBioscience™ intracellular staining kit per manufacturer’s protocol and stained for IFNγ, TNFα and IL-2. Flow cytometry was performed on a FACSymphony or LSR II (BD Biosciences); data were analyzed with FlowJo software v.10.7.2 (BD Biosciences).
Annexin 5
Manufactured by R&D Systems
Sourced in United States
Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine (PS). It is commonly used as a probe for the detection of PS exposure on the outer leaflet of the cell membrane, which is an early indicator of apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (2 mentions)
Zombie aqua fixable viability dye, by BioLegend (2 mentions)
Ebioscience intracellular staining kit, by Thermo Fisher Scientific (2 mentions)
Facsymphony, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Annexin 5, by BD (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
X rad 320, by Precision X-Ray (1 mentions)
Gm130, by Novus Biologicals (1 mentions)
Tsg101, by Novus Biologicals (1 mentions)
8 protocols using annexin 5
1
Multiparametric Flow Cytometry Analysis
2
Multiparameter Flow Cytometry Analysis
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For proliferation measurements, cells were first stained with Zombie aqua fixable viability dye (Biolegend) for 15 minutes, then surface antibodies for 20 min, then washed and fixed in 1% paraformaldehyde before flow cytometric analysis. For apoptosis assessment of PBMC and T cells, following surface staining, cells were stained with Annexin V (R&D Systems) per manufacturer's instructions and were run immediately without fixation or were fixed in 1% PFA diluted in Annexin V binding buffer, containing CaCl2 to preserve Annexin V binding. To measure intracellular cytokines, cells were treated with GolgiPlug™ (BD BioScience) for the last 5 hours of culture, then stained with fixable viability dye and surface antibodies as above. Cells were then permeabilized using Invitrogen eBioscience™ intracellular staining kit per manufacturer's protocol and stained for IFNg, TNFa and IL-2. Cells were run on a BD LSR II or BD Symphony and analyzed using FlowJo (BD Biosciences).
3
Quantifying Apoptosis by Flow Cytometry
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For flow cytometry, cells were stained with APC-conjugated Annexin V (Tonbo Biosciences, 20-6409-T025) and DAPI in 1X Annexin V binding buffer according to manufacturer instructions (R&D Systems, 4830-01-K). Fluorescence was detected and quantified on a BD LSRFortessa X-20 flow cytometer. Cells treated with 1 µM staurosporine for 4 h were used as the positive control for apoptosis. For western blot and IHC detection of apoptosis, rabbit anti-cleaved caspase 3 antibody (CST, 9661) was used.
4
Apoptosis Detection by Flow Cytometry
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Cells were fixed in 1 U/ml of RNase A (DNase free) and 10 µg/ml propidium iodide overnight in the dark at room temperature. To assess whether apoptosis had occurred, the cells were incubated with annexin-V (R&D Systems). A FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) was used to determine the number of apoptotic cells, i.e., cells with sub-G1 DNA that were annexin-V+.
5
Radiation Sensitivity of MEFs
6
Apoptosis Assay for PLFs
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WT or BMP4+/ − PLFs were plated in a 6-well plate and treated with TGF-β1 and/or BMP4 for 48 h. Apoptotic PLFs were measured using Annexin V, fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (R&D Systems, Minneapolis, MN, USA).
7
Apoptosis Detection by Flow Cytometry
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A flow cytometric apoptosis detection kit (Becton Dickinson, McKinley, MN, USA) was used according to the manufacturers’ instructions. Cell lines SCC9 and SCC25 were double-stained with 10 µL fluorescein isothiocyanate-labeled Annexin-V (R&D Systems, Minneapolis, MN, USA), to detect phosphatidylserine expression on cells during the early apoptotic phases, and with 10 µL 7-amino-actinomycin (7-AAD), to define necrotic cells. Cells positive only for Annexin-V were defined as apoptotic; cells positive for both, Annexin-V and 7-AAD, were identified as necrotic or late apoptotic cells.
8
Characterizing Amniotic Fluid Extracellular Vesicles
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Unprocessed (raw) AF lysate was prepared by centrifuging unprocessed amniotic fluid at 1000g for 10 min to pellet cell debris and proteins. The pellet was lysed in 0.2mL RIPA buffer and centrifuged at 14,000g for 10 minutes. Protein was measured with a BCA assay for both raw AF lysate and AF-EV samples and the same amount of protein (80 ug/mL) was loaded for each sample. Capillary western was performed using 12-230 kDa prefilled plates (Bio-techne) with immunoassay and total protein detection. For total protein analysis a total protein detection module for chemiluminescence based assays (Bio-techne) was used. Separation time was 25 minutes, separation voltage was 375 V, primary antibody incubation time was 40 minutes, and secondary antibody incubation time was 30 minutes. Antibody signal was detected with HDR Chemiluminescence. Antibodies used were CD9 (Cell signaling technologies), TSG101 (Novus Biologicals), Annexin V (R&D Systems), and GM130 (Novus Biologicals).
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