Cells were washed with PBS and lysed in an RIPA buffer (Sigma-Aldrich; Merck KGaA). Protein concentration was determined using the bicinchoninic acid (BCA) method of protein determination with a Microplate Manager V6 (Bio-Rad Laboratories, Inc.). Proteins (20 µg) were separated on 8–10% SDS-PAGE gel and then transferred to PVDF membranes (EMD Millipore). Membranes were blocked in 5% non-fat milk in PBST for 1 h at room temperature, and then incubated with diluted primary antibodies against GAPDH (dilution 1:1,000; cat. no. sc-166574; Santa Cruz Biotechnology, Inc.), TR4 (dilution 1:500; product code ab109513; Abcam), OCT4 (dilution 1:500; product no. 2750; Cell Signaling Technology, Inc.) overnight at 4°C. The blots were then incubated with HRP-conjugated secondary antibody (goat anti mouse; 1:10,000; cat. no. A16078; or goat anti rabbit; 1:10,000; cat. no. A16110; both from Thermo Fisher Scientific, Inc.) for 1 h at room temperature, washed, and developed in an ECL system (Bio-Rad Laboratories, Inc.).
Microplate manager v6
Manufactured by Bio-Rad
Sourced in United States, Japan
Microplate Manager v6.0 is a software application designed for the management and analysis of microplate data. It provides tools for data collection, organization, and reporting from various microplate-based experiments.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using microplate manager v6
1
Western Blot Analysis of Stem Cell Markers
2
Flow Cytometry and ELISA Data Analysis
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Flow cytometry data analysis was performed with FlowJo V.10.7.1; flow cytometry data are presented as percent of cells, median fluorescent intensity (MFI), or fold change in MFI. ELISA data were analyzed using MicroPlate Manager V.6 (BioRad). Graphs for flow cytometry, ELISA, and luminescence data were generated and statistics were performed using GraphPad Prism V.8.3. Heatmaps were generated using the ComplexHeatmap V.2.8.0 and the ggplot2 package from RStudio V.3.3.3.28 29 (link)
Fold changes, reflecting differences in protein expression and qPCR analysis, were logarithmically transformed, after which a one-sample t-test was performed. Differences in MFI reflecting protein surface expression, T cell activation, cytokine secretion levels, and cytotoxic capacity of cells, and differences in expression of immune signature genes were analyzed with the non-parametric Mann-Whitney U test between separate groups. P values of <0.05 were considered significant.
Fold changes, reflecting differences in protein expression and qPCR analysis, were logarithmically transformed, after which a one-sample t-test was performed. Differences in MFI reflecting protein surface expression, T cell activation, cytokine secretion levels, and cytotoxic capacity of cells, and differences in expression of immune signature genes were analyzed with the non-parametric Mann-Whitney U test between separate groups. P values of <0.05 were considered significant.
3
Quantifying Secreted Cytokines from GECS
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To evaluate SDF-1α, MCP-1, and IGF-1 levels secreted from GECS-conditioned medium (GECS CM), 1052 GECS cells/mm2 were plated on poly-HEMA-coated 6-well plates. After 48 h of culture, SDF-1α, MCP-1, and IGF-1 supernatant levels were determined by Mouse CXCL12/SDF-1α, Mouse CCL2/JE/MCP-1, and Mouse IGF-1 immunoassay kits (R&D Systems, Minneapolis, MN, USA). The ELISA reaction product was quantified by measuring absorbance at 450 nm and 540 nm using an iMark microplate reader, and data were analyzed using Microplate Manager v. 6.0 (both from Bio-Rad, Hercules, CA, USA).
4
Sepin-1 Inhibitory Effect on Viability
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Cells were seeded into 96‐well plates (3000 cells/well), and viability was determined using Cell Counting Kit‐8 (CCK8; Dojindo). At each time point, cells were incubated with CCK8 for 2 hours, and the absorbance was measured using Microplate Manager v6.0 (Bio‐Rad). For assessing the Sepin‐1 inhibitory effect, 24 hours after seeding, the medium was changed to medium containing Sepin‐1 (for each concentration) or dimethyl sulfoxide (0.5%; vehicle control). The IC50, which is defined as the drug concentration at which cell viability is reduced by half, was determined by GraphPad Prism 7 (GraphPad Software) as described previously.20 (link) Three sets of independent experiments were performed for each time point.
5
Evaluating Combination Therapy Efficacy
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Cells were seeded into 96‐well plates (1500 cells/well), and at each time point, cells were incubated with Cell Counting Kit‐8 (CCK8; Dojindo, Kumamoto, Japan) for 1.5 h, then the absorbance was measured using Microplate Manager v6.0 (Bio‐Rad). To evaluate the inhibitory effect of JPH203, 24 h after seeding, the cells were treated with the inhibitors at the indicated concentrations or the same amount of dimethyl sulfoxide (DMSO; 0.5%; vehicle control). The half‐maximal inhibitory concentration (IC50) definition and measurements were as previously reported.
8 (link)
To examine the effects of JPH203 in combination with a cyclin‐dependent kinase (CDK) inhibitor (dinaciclib, flavopiridol, or milciclib), combination assays were performed using a fixed concentration of JPH203 and three concentrations of each CDK inhibitor based on previous literature.
15 (link) Of the three concentrations tested, only the one that had the largest effect in combination with JPH203 is shown in the results.
8 (link)
To examine the effects of JPH203 in combination with a cyclin‐dependent kinase (CDK) inhibitor (dinaciclib, flavopiridol, or milciclib), combination assays were performed using a fixed concentration of JPH203 and three concentrations of each CDK inhibitor based on previous literature.
15 (link) Of the three concentrations tested, only the one that had the largest effect in combination with JPH203 is shown in the results.
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