Anti cd14 percp

Flow cytometric analysis of surface marker expression was performed on a FACS Canto II flow cytometer (Becton Dickinson) using a five-color setup. Anti CD206 FITC, Anti CD163 PE and Anti CD14 PerCP were bought from Becton-Dickinson. Anti-CCR7 APC was supplied by R&D systems and Anti CD11c PC7 by IOT/Beckman-Coulter.

Collected 200 μl BM specimens from MM patients and HC, respectively. Subsequently, labeling the specimens with anti‐CD138‐Bv421, anti‐CD38‐APC, anti‐CSF1‐APC, anti‐CD14‐PerCP, anti‐CD68‐FITC, and anti‐CSF1R‐Bv421 antibodies was done (BD Biosciences). Among these, CD68 is an intracellular marker. The marker of intracellular staining was fixed and permeated with IntraSure Kit (BD Biosciences). Eventually, the data of CSF1 and CSF1R were acquired by flow cytometry (FCM) (Beckman CytoFLEX).

Flow cytometry was performed using a FACSCalibur Cytometer (BD Biosciences); a phenotyping kit (MSC phenotyping kit, Miltenyi) was used to characterize the tMSCs. The following antibodies were used: anti-CD34PerCP, anti-CD45PerCP, anti-CD20PerCP, anti-CD14PerCP, anti-CD73APC, anti-CD9FITC, and anti-CD105PE (BD Pharmingen). Matched isotype controls were applied to determine background fluorescence levels.

HLA-A2 positive apheresis products were obtained from the Blood Donor Facility at the Cedars-Sinai Medical Center or purchased from HemaCare Corp. (Van Nuys, CA). Using the Elutra Cell Separation System (Terumo BCT), both monocytes and lymphocytes were isolated and red blood cells (RBCs) were removed. By staining the lymphocyte fraction with anti-CD3 antibody (BD Bioscience, Cat.564810) purification of T cells was assessed (T cells > 80%) and cells were frozen and stored in liquid nitrogen tank for later use. The monocyte fraction was stained with anti-CD14-PerCP (BD Bioscience, Cat.340585), anti-CD45-FITC (BD Bioscience, Cat.555482) and anti-CD66-PE (BD Bioscience, Cat.551480) antibodies. In order to generate DCs, more than 60% of the monocyte population needs to be CD14⁺/CD45⁺ while CD66⁺ cells are less than 10%.

One million freshly isolated PBMCs were used for cell surface marker staining in two separate 4 cc polystyrene tubes (BD, USA), where one of the tubes contained anti-CD14 PerCP (BD, USA), CD16 APC-H7, PDL1 PE (Biolegend, Germany) and the other tube contained IgG2b isotype control for PDL1 antibody along with the aforementioned fluorochrome conjugated anti-CD14 and anti-CD16. The cells were stained for 20 minutes in the dark at room temperature, washed with FACS buffer (PBS containing 1mM EDTA and 0.1% bovine serum albumin), and the resuspended pellet fixed with 0.5 ml 4% paraformaldehyde for 20 minutes. Finally, cells were washed, re-suspended in FACS buffer and data was acquired using a FACSCanto II cytometer with FACSDiva software (BD, USA), and analyzed using Flowjo 9.4.6 Software (FlowJo, USA). We included an internal quality control consisting of PBMC from a healthy control which were stained, fixed, frozen in replicate aliquots and thawed periodically as a reference to guide appropriate gating. Samples resulting in fewer than 1700 acquired monocytes and those samples with clearly suboptimal staining were excluded from analysis.

Surface BST2 expression was detected on different subsets of leukocytes, defined by anti-CD3-Alexa700, anti-CD4-Pacific Blue, anti-CD8-V500, anti-CD14-PerCP (all from BD Biosciences, Heidelberg, Germany) and anti-CD45-FITC (Miltenyi, Bergisch-Gladbach, Germany), by using an anti-BST2-APC conjugated antibody (RS38E, Biolegend). Labeled cells were fixed with 3 % formalin and analyzed on a BD LSRII flow cytometer (BD Biosciences, Heidelberg, Germany). The data files were evaluated using FlowJo Version 8.7 (Tree Star, Ashland, USA). Median fluorescence intensity (MFI) for granulocytes, monocytes and lymphocytes were determined.