Anti human cd8 pe cy7

For surface marker staining, PBMCs were labeled with the following mAbs: anti-human CD14 PE-Cy7, anti-human CD33 PE, anti-human CD11b FITC, anti-human PD-L1 PerCp-eFluor (eBioscience), anti-human HLA-DR APC, anti-human CD8 PE-Cy7, anti-human CD4 PE (BD Biosciences), anti-human CD3 PB, anti-human CD15 BV421 (Biolegend). After incubation for 20 min at RT, the cells were analyzed using flow cytometer. For whole blood staining, 100 μl of fresh whole blood was labeled with above-mentioned antibodies for 20 min at RT, then lysed with red blood cell (RBC) lysis buffer (BD Biosciences), and subjected to flow cytometry.
For intracellular staining, the cells were fixed and permeabilized using Cytofix/Cytoperm Plus kit (BD Biosciences), and stained with the corresponding intracellular Ab, anti-human IFN-γ APC (BD Biosciences), anti-human IDO PerCp-eFluor (eBioscience), anti-human IL-10 BV421 and anti-human Arg1 PE (Biolegend). Data acquisition and analysis were performed by flow cytometer. Controls for each experiment included cells that were single stained for surface markers or intracellular proteins, unstained cells, and isotype-matched antibodies.

The flow cytometry antibodies used in this study were as follows: anti-human CD3-APC-Cy7 (BD, 557832); anti-human CD4-BV510 (BD, 562970); anti-human CD8-PE-Cy7 (BD, 557746); anti-human CD45RA-BV605 (BD, 562886); anti-human CCR7-APC-R700 (BD, 565867); anti-human-CD8-APC (BioLegend, 300912); anti-human-CD25-PE (BioLegend, 302605); anti-human CD26-PE (BioLegend, 302705); anti-human CD49f-FITC (BioLegend, 313605); and the fluorescent dye 7-AAD (BioLegend, 420404). The cells were incubated with 1% Fc receptor blocker for 10 minutes and incubated with antibodies for 30 minutes. Flow cytometry analyses were conducted using the BD FACS Canto system. The data were analyzed with FlowJo software.

hESC/OP9 and iPSC/OP9 cell cocultures were harvested and washed with FACS buffer (PBS with 2% FBS). Cells were analysed for HSC differentiation efficiency using an anti-human CD34-PE-Cy7 monoclonal antibody (BD). Mouse peripheral blood cells and femur BM cells were collected to analyse the human cell ratios. The cells were treated with erythrocyte lysate (E Bioscience), washed with FACS buffer and stained with the following monoclonal antibodies from BD Biosciences: anti-human CD3-FITC, anti-human CD8-PE-Cy7, anti-human CD31-PE, anti-human CD34-PE-Cy7, anti-human CD43-FITC, anti-human CD45-APC and anti-human CD71-PE. Staining of erythrocytes with anti-human CD235-APC monoclonal antibody (BD Biosciences) does not require lysis. Finally, the cells were resuspended in 200 μl of FACS buffer and analysed by flow cytometry on a weekly basis for 10 weeks after transplantation.