Pe anti human cd45 clone 2d1

FLS were isolated from synovial membranes as described [39 (link)]. FLS were propagated until Passage 4 in a humidified atmosphere at 37 °C with 10% CO₂ using Dulbecco’s Modified Eagle’s Medium (DMEM) that was supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES buffer, 100 U/mL penicillin, and 0.1 mg/mL streptomycin, 1.0 g/L glucose, and 584 mg/mL L-glutamine.
The purity of FLS was determined prior to treatment on a FACSCanto II (BD Biosciences, Heidelberg, Germany), confirming the presence of at least 90% (96.9 ± 3.2) of cells that stained positively for the fibroblast antigen CD90 using APC anti-human CD90 (clone 5E10, RRID: AB_893440, BioLegend, London, UK). Hematopoietic cells were negative against PE anti-human CD45 (clone 2D1, RRID: AB_2566369, BioLegend). The media were consistently tested negative for contamination with mycoplasma using the PCR Mycoplasma Test Kit I/C (PromoCell, Heidelberg, Germany).

The purity of FLS was determined at the end of passage 4–5 with a BD FACSCANTO II flow cytometer (Becton Dickinson, Heidelberg, Germany). After trypsinization, cells were stained with APC anti-human CD90 (clone 5E10) and PE anti-human CD45 (clone 2D1) or APC mouse IgG1 and PE mouse IgG1 antibodies (clone MOPC-21, BioLegend, San Diego, CA, USA). More than 80% of cells used in the experiments were stained positively for the fibroblast-specific antigen CD90 (87.1 ± 18.4%), whereas staining for CD45 was negative.

The purity of FLS was determined at the end of passage 5 using a BD FACSCANTO II flow cytometer (Becton Dickinson, Heidelberg, Germany). After trypsinization, cells were stained with APC anti-human CD90 (clone 5E10) and PE anti-human CD45 (clone 2D1) or APC mouse IgG1 (clone MOPC-21) and PE mouse IgG1 (clone MOPC-21) antibodies (BioLegend, San Diego, CA, USA). More than 90% of the cells used in the experiments stained positively for the fibroblast-specific antigen CD90 (96.4 ± 3.5%), whereas staining for CD45 was negative.