Apoptosis was analyzed using an Annexin V assay kit (BD Biosciences Pharmingen, San Jose, CA, USA). Briefly, HEI-193 cells were seeded at 3 × 105 cells/well in 6-well plates. After 24 h, the medium was replaced with serum-free medium and cells were incubated with 10–20 μM of DL-SFN for 48 h. Both floating and attached cells were then collected by trypsinization. After centrifugation at 1,300 rpm for 3 min, the cells were washed twice with PBS and incubated in 1 × binding buffer (10 m HEPES/NaOH (Ph 7.4), 140 mM NaCl, and 2.5 mM CaCl2) containing annexin V-fluorescein isothiocyanate (FITC) and propodium iodide (PI) at room temperature for 10 min in the dark. Stained cells were then analyzed using a FACScan flow cytometer (Becton, Franklin Lakes, NJ, USA) within 1 h. The data were analyzed using WinList 5.0 software (Verity Software House, Topsham, ME, USA).
Winlist 5
Manufactured by Verity Software House
Sourced in United States
WinList 5.0 is a software application designed for flow cytometry data analysis. It provides users with tools to visualize, analyze, and interpret flow cytometry data. The software supports a range of features, including multi-parameter data display, gating, and statistical analysis.
Automatically generated - may contain errors
Lab products found in correlation
Facscan flow cytometer, by BD (2 mentions)
Annexin 5 assay kit, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Live dead fixable blue stain kit, by Thermo Fisher Scientific (1 mentions)
Streptavidin pe, by Jackson ImmunoResearch (1 mentions)
Enzyme free cell dissociation solution, by Merck Group (1 mentions)
5 protocols using winlist 5
1
Annexin V Assay for Apoptosis
2
Flow Cytometry Analysis of Cardiac Fibroblasts
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Flow cytometry was performed as previously described.10 (link) Briefly, primary cardiac fibroblasts (CD105+CD31−CD45−) from passage 1 were cultured in 10% FBS DMEM in the absence or presence of 30 ng/mL of Neuregulin‐1β for 48 hours. Cells were trypsinized, washed with PBS and resuspended in PBS/0.5% BSA/2 mmol/L EDTA buffer containing murine Fc block reagent (BD Biosciences). The cells were then incubated with CD105‐APC (clone MJ7/19; Biolegend) antibody for 20 minutes at 4°C, washed once with 10 volumes of cold PBS/BSA/EDTA, fixed and permeabilized using 4% paraformaldehyde and 0.1% saponin in Ca/Mg free DPBS. Cells were stained with fluorescein isothiocyanate (FITC)‐conjugated anti‐αSMA (Clone 1A4, Sigma) antibody for 25 minutes at 4°C. Mouse FITC‐conjugate IgG2a (Clone UPC‐10, Sigma) were used as an isotype control. Viable and nonviable cells were distinguished using LIVE/DEAD Fixable Blue Stain kit (Invitrogen). Data acquisition was performed using an LSRII flow cytometer (BD Biosciences), and the data were analyzed with WinList 5.0 software (Verity Software House, Inc). Antigen negativity was defined as having the same fluorescent intensity as the isotype control.
3
Cell Cycle Arrest Analysis by SFN
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HEI-193 cells were used to study cell cycle arrest caused by SFN. HEI-193 cells were seeded in 6-well plates at 5 × 105cells/well. After SFN application for 48 h, the floating cells and attached cells were collected by trypsinization, centrifuged at 1200 rpm for 5 min, fixed with ice-cold 70% ethanol, and stored at 4 °C for 1 h. Fixed cells were resuspended with PBS containing RNase A (100 ug/ml, Intron, Daejeon, Korea) for 30 min at 37 °C, and a propodium iodide (PI) solution (20 ug/ml, Invitrogen) was added. Stained cells were analyzed using a FACScan flow cytometer (Becton) and WinList 5.0 software(Verity Software House).
4
Dual Staining for Cell Viability Analysis
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Samples were run by flow cytometry (FCM) in a Becton–Dickinson FASort model equipped with an argon laser for excitation at 488 nm and 15 mW and filters at 525 and 630 nm. Samples were adjusted to an event rate of 800–2000 cells s−1 and a total of 10,000 events were registered per sample. To determine cell viability by FCM, double staining was performed accordingly to (Hewitt et al. 1999 (link)). PI and BOX were used for viability studies on living cells. The FCM probe fluoresceinpropidium iodide (PI) was purchased from Sigma–Aldrich, while bis-(1,3-dibutylbarbituric acid) trimethine oxonol (Bis-oxonol, BOX) was purchased from Molecular Probes Inc. Stained cells were diluted in phosphate buffered saline solution pH 7.2 (PBS). FALS and RALS values allowed cell debris discrimination and a total of 10,000 events were used for statistical data analysis.
Heat stressed cells treated at 60 °C for 30 min and exponentially growing cells were used as positive and negative controls, respectively. The green fluorescence channel for BOX-stained cells (X-axis) was plotted versus the red fluorescence channel for PI stained cells (Y-axis). Flow cytometry data were analysed with WinList 5.0 (Verity Software House) software.
Heat stressed cells treated at 60 °C for 30 min and exponentially growing cells were used as positive and negative controls, respectively. The green fluorescence channel for BOX-stained cells (X-axis) was plotted versus the red fluorescence channel for PI stained cells (Y-axis). Flow cytometry data were analysed with WinList 5.0 (Verity Software House) software.
5
Measuring BAFF Binding to TNFR Superfamily Receptors
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BAFF was biotinylated using Pierce NHS-LC-biotin (Thermo Fisher Scientific); HEK293 cells were stably transfected with human BR3, human TACI, or human BCMA. Expression was confirmed with specific antibody binding and flow cytometry. The cells were removed from the cell culture flask using Enzyme Free Cell Dissociation solution (EMD Millipore), resuspended at 106 cells/mL in FACS buffer (D-PBS, 2% FBS, 0.1% sodium azide) with 1 mg/mL goat IgG added. For cells expressing BCMA, 300 ng/mL biotinylated BAFF + 50 μg/mL tabalumab were pre-incubated for 15 minutes at RT. For BR3, TACI, or vector control cells, 18.8 ng/mL biotinylated BAFF + 5 μg/mL tabalumab were pre-incubated for 15 minutes at RT. The mixture was added to cells and incubated for 20 minutes on ice. The cells were washed with FACS buffer and resuspended with streptavidin-PE (phycoerythrin; Jackson ImmunoResearch) at a 1:150 dilution and incubated on ice for 15 minutes. The cells were washed as above and then resuspended in FACS buffer. The fluorescence was measured using a Guava EasyCyte Plus instrument (EMD Millipore) counting 2,000 cells with the Express Plus protocol. The files were converted to FCS files and the overlays were generated using WinList 5.0 (Verity Software House, Topsham, ME, USA).
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