Cd11b c

Erythrocytes were lysed using 1× PharmLyse (BD Biosciences Franklin Lanes, NJ, USA) according to the manufacturer’s instructions. Cells were washed and resuspended in PBS pH 7.4, containing 2% FBS and 0.09% sodium azide (stain buffer). An amount of 5 × 10⁵ cells were stained for 30 min on ice with immune cell-specific antibodies (BD Biosciences, Franklin Lakes, NJ, USA) as follows: CD8a (#561611), CD4 (#554837), CD3 (#554833), CD11b/c (#743980), CD161a (#555009), or CD45RA (#554881) at a concentration of 1:100 diluted in stain buffer. Cells were washed two times with 2 mL stain buffer and centrifuged at 350× g for 5 min at 4 °C. Cells were resuspended in 400 μL of stain buffer and immediately analyzed using a BD FACSymphony A3 Flow Cytometer (BD Biosciences, Franklin Lanes, NJ, USA) at the UMMC Flow Cytometry and Cell Sorting Core Facility. Data were analyzed using FlowJo software version 10.8.2. Gating strategy for both the brain and PBL are included in Supplemental Figure S1.

Biodistribution of labeled AD-MSCs (2×10⁶) with DiI after IV administration was analyzed using immunofluorescence techniques in treated animals at 14 d after administration. Cryosections (10 μm thick) of brain, kidney, liver, lung and spleen were labeled with 4’,6-diamidino-2-phenylindole (DAPI) and CD11b/c (monoclonal antibody diluted 1:300, BD Biosciences) and were analyzed by immunofluorescence staining.
Moreover, tumor formation and the presence of infiltrating cells were analyzed in the brain and the peripheral organs at three months after AD-MSCs IV administration in five treated animals using gross macroscopic study and hematoxylin and eosin (H&E) staining.