Anti gcsf

Crohn’s disease (n = 5) or GvHD (n = 5) MSCs were seeded into 96-well plates at a density of 50,000, 25,000, 12,500, or 0 cells per well, and 100,000 human PBMCs (n = 3) were added to each well. 1000 ng/mL SEB (Toxin Technology, Sarasota, FL) was used to activate T cells. MSCs at a similar density without PBMCs were used as a reference control. To test the effect of freezing/thawing on MSC fitness, thawed MSCs from cryopreservation were used in similar assays. Thawed and fresh MSC viability count was determined by mixing equal volumes of 0.4% trypan blue and cell mixture and analyzed either using a he-macytometer or by automated cell counting (Invitrogen Countess, USA). Both fresh and thawed cell concentrations were normalized based on the viability count. Subsequently, they were seeded into 96-well plates as above, and 1,000 ng/mL SEB and PBMCs were added immediately. For blocking experiments, anti-VEGF (Thermo Fisher, USA), anti-GCSF, anti-CXCR3, anti-IFNαβR1, anti-CCL2, and anti-IL-7 or isotype control antibodies (R&D Systems, USA) were added to MSC and SEB-activated PBMC co-cultures at concentrations of 25 μg/mL. Four days after culture, a Ki67 flow cytometric proliferation assay was performed according to the manufacturer’s instructions with CD3-APC-Cy7 and Ki67-PE antibodies (BD Biosciences, San Jose, CA).