ID8 and SKOV3 cells were plated in a 75-cm2 dish (1.5×104 cells/ml) and allowed to adhere overnight at 37°C prior to treatment. Following treatment with the specified concentrations of MK1775, the cell apoptosis assay was conducted using cell staining with annexin V (2.5 µg/ml) and 7-aminoactinomycin D (7-AAD) (1 µg/ml) for 15 min at room temperature (25°C) in the dark, according to the manufacturers protocol. The apoptosis assay was performed 24 or 48 h after drug treatment using MACS Quant Analyzer (Miltenyi Biotec, Cologne, Germany). The data were analyzed using Flow Jo software version 10.0.7r2 (Tree Star, Inc., Ashland, OR, USA). Cells that stained negative for annexin V-allophycocyanin and 7-AAD were classified as not undergoing determinable apoptosis, double-positive cells were classified as late-state apoptosis, single-positive cells for annexin-V were classified as early apoptosis and single-positive cells for 7-AAD were classified as dead or necrotic cells.
Flow jo software version 10.0.7r2
Manufactured by Tree Star
Sourced in United States
FlowJo software version 10.0.7r2 is a data analysis software for flow cytometry data. It provides tools for data visualization, gating, and statistical analysis.
Automatically generated - may contain errors
2 protocols using flow jo software version 10.0.7r2
1
Apoptosis Assay for MK1775 Treatment
2
Detecting SARS-CoV-2-Specific B Cells
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In order to detect SARS-CoV-2-specific B cells, biotin-labeled SARS-CoV-2 spike RBD protein (40592-V08H2-B; Sino Biological, Beijing, China) was mixed with Streptavidin BV421-11 (405225; Biolegend, California, CA, USA) at a molar ratio of 4:1 for 1 h to obtain an antigen probe. According to the manufacturer's instructions, peripheral blood mononuclear cells were isolated from whole heparinized blood by Histopaque (10771; Sigma-Aldrich, St Louis, MO, USA) density gradient centrifugation. After washing with flow cytometry staining (FACS) buffer (phosphate-buffered saline with 2% fetal bovine serum), staining was performed for 30 min at 4°C with an antigen probe (1:33.3) and the following binding antibodies at 1:50 dilution: anti-human CD3 (300430), anti-human CD19 (302212), anti-human CD21 (354918), and anti-human CD27 (356406) all purchased from Biolegend. After staining, the cells were re-washed and suspended in 200 µL FACS buffer. Samples were evaluated using a CytoFLEX cytometer (Beckman Coulter, Inc., Brea, CA, USA), and FlowJo software version 10.0.7r2 (Treestar Inc., Ashland, OR, USA) was used for data analysis.
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