Dulbeco’s modified Eagle’s medium (DMEM) was purchased from Sigma (St. Louis MO), supplemented with 10% FBS (Sigma). Penicillin, streptomycin, L-glutamine and Hepes was from Lonza (Basel, Switzerland). Antibodies to Nanog and β-actin were from Abcam (Cambridge, UK). Mitotracker Green and Mitosox Red were purchased from Molecular Probes, Invitrogen (Carlsbad, CA). TOPRO-3, DAPI (4’,6-diamino-2-phenylindole dihydrochloride) and ProLong Antifade reagent were from Invitrogen. The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit was from Invitrogen and EpiSeeker hydroxymethylated DNA Quantification Kit from Abcam. Kits for molecular studies were purchased from Applied Biosystems, Life Technologies (Paisley, UK). DIO rodent purified HFD (“Diet induced Obesity” with 60% energy from fat, formula 58Y1) was obtained from TestDiet (IPS Product Supplies Ltd, London, UK). Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich.
Violet ratiometric membrane asymmetry probe dead cell apoptosis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Netherlands
The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis kit is a fluorescence-based assay designed to detect apoptosis, or programmed cell death, in cells. The kit utilizes a ratiometric probe that binds to phosphatidylserine, a lipid that translocates to the outer membrane during apoptosis. The probe generates a fluorescent signal that can be measured to quantify the extent of apoptosis in a sample.
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Lab products found in correlation
Hm40 3, by Thermo Fisher Scientific (2 mentions)
Mitoprobe diic1 5 kit, by Thermo Fisher Scientific (2 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Episeeker hydroxymethylated dna quantification kit, by Abcam (1 mentions)
Dulbeco s modified eagle s medium dmem, by Merck Group (1 mentions)
4 6 diamino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Prolong antifade reagent, by Thermo Fisher Scientific (1 mentions)
Mitotracker green, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Hepes, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
7 protocols using violet ratiometric membrane asymmetry probe dead cell apoptosis kit
1
Stem Cell Culture and Analysis
2
Quantifying Apoptosis by Flow Cytometry
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Apoptosis was measured using the Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit (Invitrogen) as described [19 (link)]. Both floating and adhered cells were collected and pelleted by centrifugation. Adhered cells were detached with trypsin. Cells were suspended in Hanks Balanced Salt Solution (HBSS) containing 4'-N,N-diethylamino-6-(N,N,N-dodecyl-methylamino-sulfopropyl)-methyl-3-hydroxyflavone (F2N12S, 200 nM) plus SYTOX AAdvanced dead cell stain solution (1 mM) at 106 cells/ml and incubated for 5 min at room temperature in the dark. Apoptosis measurements were performed with an LSRFortessa cytometer (BD Biosciences, San Jose, CA) at 405 nm and 488 nm. Data were analyzed with BD FACSDiva Software.
3
Evaluating Apoptosis in HRMECs
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The ratio of apoptotic cells to total cells was assessed using the violet ratiometric membrane asymmetry probe/dead cell apoptosis kit (Thermo Scientific, United States) followed by flow cytometry. HRMECs were incubated with 4’-N,N-diethylamino-6-(N,N,N-dodecyl-methylamino-sulfopropyl)-methyl-3-hydroxyflavone (F2N12S) and SYTOX AADvanced for 5 min. The data was collected on BD LSRFortessa X-20 (BD Biosciences, United States) according to the manufactory’s recommended settings. The ratio of apoptotic cells was analyzed by Flowjo 10.8.1.
4
Flow cytometry analysis of Bhlhe41 reporter B cells
5
Cell Cycle and Apoptosis Analysis
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Cells were seeded onto six-well plates at a density of 0.5 × 106 cells/well in DMEM containing 10% (v/v) FBS. After overnight incubation cells were starved of FBS (down to 2% (v/v)) and treated with recombinant protein for 24 h. Cells were then trypsinized, washed twice, and permeabilized using 70% (v/v) ethanol prior to propidium iodide staining [27 (link)]. Cells were next analyzed using a BD Biosciences FACS Calibur.
For apoptosis detection, the Violet Ratiometric Membrane Asymmetry Probe/Dead cell Apoptosis Kit (Thermo Fisher Scientific, Netherlands) was used as indicated by the manufacturer and apoptosis was analyzed using a BD Biosciences LSR-II. Single cells were gated, and the resulting DNA distributions were analyzed using the flow cytometry analysis software FlowJo.
For apoptosis detection, the Violet Ratiometric Membrane Asymmetry Probe/Dead cell Apoptosis Kit (Thermo Fisher Scientific, Netherlands) was used as indicated by the manufacturer and apoptosis was analyzed using a BD Biosciences LSR-II. Single cells were gated, and the resulting DNA distributions were analyzed using the flow cytometry analysis software FlowJo.
6
Flow cytometry analysis of Bhlhe41 reporter B cells
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For flow cytometric analysis of Bhlhe41 reporter expression, hCD2– FO B cells from Bhlhe41-iCre-IRES-hCD2 and wild-type mice were sorted by flow cytometry, seeded at a density of 5 × 105 cells/ml in the presence of 25 μg/ml LPS or 10 μg/ml anti-IgM and 20 ng/ml IL-4 or 2 μg/ml anti-CD40 antibody (eBioscience, HM40-3) and 20 ng/ml IL-4 or left unstimulated. hCD2 expression was assessed by flow cytometric analysis at days 2 and 4 of in vitro stimulation. For in vitro survival experiments, VH12/Vκ4 transgenic wild-type and DKO CD19+ cells were sorted by flow cytometry and cultured overnight in the presence or absence of IL-5 (10 ng/ml). Cell death was assessed by staining with 7-ADD followed by flow cytometric analysis. All experiments were performed in IMDM medium containing 10% fetal calf serum, 1 mM glutamine and 50 μM β-mercaptoethanol. Ex vivo cell death was assessed by using the Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis kit together the MitoProbe DiIC1(5) kit (both from ThermoFisher Scientific) according to the manufacturer’s instructions. Peritoneal cells from 10% chimeras were pre-stained with antibodies against CD19 and CD45.2 to gate on the VH12/Vκ4 transgenic donor B cells.
7
Etoposide-Induced Apoptosis Modulation
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Cells were treated for 18 h with 10 μM etoposide ± HGF. Apoptosis was assessed by flow cytometry using the Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit (Cat. no. A35137; Thermo Fisher Scientific).
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