The trypsinized GB-MSCs and their respective PBMCs were washed with PBS, centrifuged at 280 g for 10 minutes, counted, and brought to a concentration of 1.105 cells. GB-MSCs were tested for expression of the following surface markers: PD-L1-APC (BD Pharmingen, USA), CD73-PE, CD90-FITC, CD105-PerCP/Cy5-5 (eBioscience, USA), CD45-FITC/CD34-PE, CD44-FITC, CD146-PE, and HLA-A, B, C-FITC (Becton Dickinson, USA) and intracellular markers: Nestin-PE, Sox-2-PerCP, and GFAP-Alexa Fluor 488 (eBioscience, USA). For the detection of intracellularly expressed markers, the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Pharmingen, USA) was used following the manufacturer's instructions.
PBMCs from healthy volunteers were tested for surface expression of CD3-FITC, CD4-PerCP, CD161-PE, CD196-Alexa Fluor 488, CD25-FITC, CD14-FITC, CD80-PE, CD86-APC, and HLA-DR-PerCP and intracellular expression of FoxP3-PE (BD Pharmingen, USA) by using the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Pharmingen, USA). Cells were processed according to the manufacturer's instructions, fixed with CellFix (BD, USA), and analyzed by FACSCalibur flow cytometer (BD, USA). Software CellQuest and WinMDI 2 were used for further analysis.
PBMCs from healthy volunteers were tested for surface expression of CD3-FITC, CD4-PerCP, CD161-PE, CD196-Alexa Fluor 488, CD25-FITC, CD14-FITC, CD80-PE, CD86-APC, and HLA-DR-PerCP and intracellular expression of FoxP3-PE (BD Pharmingen, USA) by using the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Pharmingen, USA). Cells were processed according to the manufacturer's instructions, fixed with CellFix (BD, USA), and analyzed by FACSCalibur flow cytometer (BD, USA). Software CellQuest and WinMDI 2 were used for further analysis.