Anti cd36 pe

Flow cytometry was used to measure CD14, CD36 and CD11b on differentiated U937 cells. Differentiated U937 cells were either treated or not treated with 1000 ng·mL⁻¹ of HSPA1A or HSPB1 and incubated for 6 h. Cells were either immune‐stained with or without 5 μL Anti‐CD14: APC‐CyTM7, Anti‐CD36: PE, Anti‐CD11b: FITC (BD BioSciences, Franklin Lakes, NJ, USA). Cells were also stained with 10 μL propidium iodide staining solution (BD BioSciences) for 1 h at 4 °C in the dark. The cells were gated using flow cytometry BD Accuri™ c6 (Piscataway, NJ, USA) to select by circling viable cells. This ensured only viable cells were analysed and not cell debris.

The immunophenotypes of the BM-DFATs, SC-DFATs, BM-MSCs, ASCs at passage 2 were identified using flow cytometry as previously described [13 (link)]. The cells grown to 60% confluence were suspended at a density of 5 × 10⁵ cells per tube and incubated with various anti-human antibodies conjugated with phycoerythrin (PE) or allophycocyanin (APC). The following antibodies were used: anti-CD73-PE, anti-CD90-APC, anti-CD105-PE, anti-CD31-PE, anti-CD45-APC, anti-HLA-DR-PE, anti-CD106-PE, anti-CD54-APC, and anti-CD36-PE (all from BD Biosciences, San Jose, CA). Mouse IgG1-PE, mouse IgG1-APC, mouse IgG2a-PE, mouse IgG2b-APC, and mouse IgM-PE (all from BD Biosciences) were used as negative controls. The fluorescence intensity of the cells was evaluated by a FACSAria flow cytometer (Becton Dickinson, Bedford, NJ), and data were analyzed using FlowJo software (version 10.6.1, FlowJo, Ashland, OR). Positive cells were counted and compared with the signal of corresponding immunoglobulin isotypes. A minimum of 1 × 10⁴ events were recorded for each sample, and analysis was performed at least three separate times for each condition tested.