Cd49d apc

Cultured cells were harvested at different time intervals. For morphological characterization, cytospin smears were prepared and stained with Wright’s and Giemsa stain. A total of 500 cells were counted in random non-overlapping fields to determine the different stages of erythropoiesis process.
Phenotypic characterization was done by analyzing the expression of different cell surface markers. Cells were harvested and suspended in 50 μl of 1× phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (Sigma Aldrich, St. Louis MO, USA) and 0.1% azide (Sigma Aldrich, St. Louis MO, USA), and then incubated with specific fluorochrome-tagged antibodies for 45 min at 4 °C in the dark. Proper isotype-matched antibodies were used as controls. The stained cells were washed, re-suspended in PBS, and acquired on FACS Canto II (BD, San Jose, CA, USA). Data were analyzed by using BD FACS DIVA or BD Flow Jo software. The details of the antibodies used are as follows: CD71-FITC, CD235a-APC (ebioscience, San Diego, California, USA), CD235a-Brilliant violet, CD49d-APC, AnnexinV FITC (BD Bioscience, San Jose, CA, USA).

Migrated and non-migrated human T cells were stained with fixable viability dye (FVD) eFluor780 (#65–0865-14, eBioscience, Thermo Fisher), CD4-PE (#300508, BioLegend), CD8-PE (#21270084, ImmunoTools), CD49d-APC (#559881) and CD11a-BV650 (#745344) (all BD Biosciences, Franklin Lakes, USA). Surface expression was calculated as median fluorescence intensity (MFI). Using a classical gating strategy, cells were selected by using FSC-A and SCC-A and singlets based on the FSC-A vs. FSC-H. Dead cells were excluded by FVD eFluor780 positivity and the gate defined on the positive CD4/CD8 population (Additional file 1: Fig. S4 a, c). Measurements were performed with the BD LSRFortessa™ X-20 (BD Biosciences) and analyzed with Flow Jo v10.7.

Frozen PBMCs were thawed, and cell viability was consistently >90%. After the incubation with the FcR blocking (BD) and Aqua Dead Cell Stain (Molecular Probes), as described in Section “Flow Cytometry of BM PCs,” the cells were washed with FACS buffer and surface-stained for 15 min at 4°C with antibodies against CD3 BV786 (BD), CD20 BV421 (BD), CD138 PE (eBioscience), CD80 clone L307.4 APC-H7 (BD), HLA-DR clone L243 PerCP-Cy5.5 (BD), CD14 clone M5E2 BV786 (BD), CD16 clone 3G8 BV786 (BD), CD98 FITC (eBioscience), and CD49d APC (BD) or CD31 Alexa 647 (BD). Cells were washed twice with FACS buffer and incubated in 100 μl of Cytofix/Cytoperm (BD) for 20 min at 4°C. Cells were then washed twice in Perm/Wash buffer (BD) and intracellularly stained in 100 μl of Perm/Wash buffer for 15 min at 4°C with IgG clone G18-145 PE-Cy7 (BD), followed by two washes in Perm/Wash buffer and two in FACS buffer. All Abs were previously titrated for optimal staining of rhesus macaque PBMCs. Samples were collected on a FACS LSRII (BD Immunocytometry Systems) and analyzed using FlowJo software.