Cd14 pacific blue

Cell surface expression of PD1 (CD279) on CD4⁺T cells, CD8⁺T cells, CD56⁺ NK cells and CD3⁺CD8⁺CD56+NKT cells, and PD-L1 (CD274) on CD138+ MM cells, CD14⁺ monocytes/macrophages, CD11b⁺CD14⁺HLA-DR⁺ antigen presenting cells (APCs), CD11b⁺CD14⁻HLA-DR^−/lowCD33⁺CD15⁺ nMDSC, and CD11b⁺CD14⁺HLA-DR^−/low mMDSC was determined on PBMCs or BMMCs from MM patients or healthy donors by multiparameter flow cytometry analysis. Cells were stained with CD11b APCCy7, CD14 Pacific blue, HLA-DR PECy7, CD33 PECy5, and CD15 FITC conjugated MAbs (BD Biosciences, San Jose, CA) for MDSC; as well as CD138 APC for MM cells, and CD4 PE-Cy5, CD8 PE-Cy7, CD56 FITC for effector cells (BD Biosciences, San Jose, CA).

Cryopreserved PBMCs from each patient obtained at day 0 and day 56 were assayed for phenotypic markers consistent with NK cells, T lymphocytes, myeloid derived suppressor cells (MDSC) and T regulatory cells as previously described (26 (link), 32 (link)). Briefly, PBMC from each patient were suspended at a concentration of 1x10⁷/mL in flow staining buffer (PBS containing 1% FBS). Cells were incubated with fluorochrome-labeled antibodies for one hour at 4°C. Specific antibodies include CD4-APC (Beckman Coulter), CD8-APC (Beckman Coulter), NKRD1 (Beckman Coulter), CD33-PE (BD Biosciences), HLA-DR PERCP-Cy5.5 (eBioscience), and CD14-Pacific Blue (BD Biosciences). PBMC were also labeled with the appropriate isotype control antibodies for each fluorochrome to use as negative controls. Cells were then washed with flow buffer, fixed with 1% formalin, and stored at 4°C until analysis. All samples were run on a BD LSR II flow cytometer, and were subsequently analyzed with FlowJo software (Tree Star, Inc.). Monocytic MDSC were defined as cells with a CD33⁺HLA-DR^negCD14⁺ phenotype. T regulatory cells were defined as CD4⁺CD25⁺FoxP3⁺ and assessed using the commercially available Human T regulatory cell staining kit per manufacturer’s recommendations (eBioscience).