Polyvalent human immunoglobulin

For phenotype characterization of white blood cells, a multi-parameter analysis was carried out by flow cytometry (Navios, Beckman Coulter®). Whole blood was collected in tube field with cellular antigen stabilization solution (Transfix, Cliniscience) and kept at 4 °C up to 14 days. Red blood cells were lysed with Macs lysing solution. To minimize non-specific antibody binding, after centrifugation, cells were incubated in a buffer containing PBS, 2% human albumin (LFB), and 2 μg/mL polyvalent human immunoglobulin (R&D Systems) before staining. Cells were then incubated with fluorescent monoclonal antibodies: CD8a (OX8, eBioscience), CD4 (OX35, eBioscience), CD3 (1F4, BioRad), CD80 (3H5, BioRad), CD279 or PD-1 (KLH, Bioss), CD274 or PD-L1 (KLH, Bioss), CD11b/c (REA, Miltenyi), CD28 (REA, Miltenyi), CD45 (REA, Miltenyi), CD86 (REA, Miltenyi), and MHC2 (REA, Miltenyi), at a saturating concentration for 20 min at + 4 °C. Isotype antibodies, non-stain cells, and fluorescence minus one were used as controls. The FlowJo software was used to set up gating and analyze positivity frequencies of the makers of interest.

For MSC phenotypic characterization, a multi-parameter analysis was carried out by flow cytometry (Navios, Beckman Coulter^®, Brea, CA, USA) on cells harvested at the end of passage 1 and 2. After trypsin detachment, cells were centrifuged and incubated in a buffer containing PBS, 2% human serum albumin (LFB, Les Ulis, France) and 2 µg/mL polyvalent human immunoglobulin (R&D Systems, Minneapolis, MN, USA) to minimize non-specific antibody binding. Cells were then incubated with fluorescent monoclonal antibodies at a saturating concentration (5 µL per 10⁵ cells): CD90, CD73, CD105, CD45, HLA-DR, CD146, CD34 and isotypic controls (Iotest, Beckman Coulter, Brea, California, USA) for 20 min at 4 °C. After 2 PBS washes, analyzes were carried out by flow cytometry (Navios, Beckman Coulter^®). The FlowJo software was used to analyze 10,000 stained cells. The percentage of positive stained cells was calculated in comparison to the isotypic control after gating on FS/SS parameters.