All cell surface staining and washing steps were performed in PBS containing 1% BSA (w/v). Cells were incubated with specific mouse monoclonal antibodies (mAbs) for 15 min at 4 °C. The following mAbs were used for flow cytometry: FITC-conjugated CD1a (DakoCytomation, Glostrup, Denmark), CD34, CD86, and Sorafenib in vitro HLA-DR (BD Biosciences, San Diego, CA), PE-conjugated CD14 (DakoCytomation), CD54 and CD80 (BD Biosciences). Mouse IgG1, conjugated to FITC or PE were used as isotype controls (BD Biosciences) and propidium iodide (PI) (BD Biosciences)
was used to assess cell viability. FACSDiva software was used for data acquisition with FACSCanto II instrument (BD Bioscience). 10,000 events were acquired, gates were set based on light scatter properties to exclude debris and non-viable cells, and quadrants were set according to the signals from isotype controls. Further data analysis was
performed, using FCS Express V3 (De Novo Software, Los Angeles, CA). All chemicals should be stored according to instructions from the supplier, in order to ensure stability of compounds. Chemicals should be dissolved in water when possible or DMSO for hydrophobic compounds. As many chemicals see more will have a toxic effect on the MUTZ-3 cells, this toxicity needs to be monitored. Some chemicals are poorly dissolved in cell media; therefore the maximum soluble concentration needs to be assessed as well. The chemical that is to be tested should be titrated to concentrations ranging from 1 μM to the maximum soluble concentration in cell media. For freely soluble compounds, 500 μM should be the upper end of the titration Rebamipide range. For cell stimulations, chemicals should be dissolved in its appropriate solvent as 1000× stocks of target in-well concentration, called stock A. A 10× stock, called stock B, is prepared by taking 10 μl of stock A to 990 μl of cell media. 200 μl of stock B is then added to the wells containing 1.8 ml seeded cells. For the samples dissolved in DMSO, the in-well concentration of DMSO will thus be 0.1%. Following incubation for
24 h at 37 °C and 5% CO2, harvested cells are stained with PI and analyzed with a flow cytometer. The relative viability of cells stimulated with each concentration in the titration range are calculated as Relative vialbility=fraction of viable stimulated cellsfraction of viable unstimulated cells·100 For toxic compounds, the concentration yielding 90% relative viability (Rv90) should be used for the GARD assay. For non-toxic compounds, a concentration of 500 μM should be used if possible. For non-toxic compounds that are insoluble at 500 μM in cell media, the highest soluble concentration should be used. Whichever of these three criteria is met, only one concentration will be used for the genomic assay. The concentration to be used for any given chemical is termed the ‘GARD input concentration’.
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