Anti cd45 pe cy7

Flow cytometry was used to identify the proportions of the different cell types contained in the SVF, as previously described [14 (link)]. Cells (350000) were stained with anti-CD3 FITC (T cells; BD; #559975), anti-CD11b FITC (myeloid cells; BD; #561684), anti-CD45 PE/Cy7 (peripheral leukocytes; BD; #561588), anti-rat macrophage subset PE (BD; #554901), or anti-rat granulocytes FITC (BD; #554907) 30 min prior to analysis. Primary anti-CD36 (Abcam; #ab23680), secondary goat anti-mouse IgG (H + L) Alexa Fluor 594 (Thermo Fisher; #A-11005), and anti-CD45 PE/Cy7 (BD; #561588) antibodies were used to identify adipocytes. Primary anti-CD34 (R&D Systems; #AF6518-SP), secondary donkey anti-sheep IgG (H + L) PerCP (R&D Systems; #F0128), and anti-CD31 PE (BD; #555027) antibodies were used to identify lymphohematopoietic cells, endothelial cells, smooth muscle cells, and adipose-derived stem cells. Samples were run on a BD LSR II Flow Cytometer (BD). Ten thousand events were collected, and data was analyzed using the FACSDiva software (BD).

Tumor cells obtained from control or treated mice were thawed, centrifuged, and stained in one-step test using monoclonal antibodies conjugated with fluorophores: anti-CD45 PE-Cy7, anti-CD4 APC, anti-CD8 FITC, and anti-CD49b PE (all from BD Biosciences). In order to eliminate dead cells during the analysis, cells were additionally stained with DAPI dye (Molecular Probes). The analysis was carried out using LSR Fortesssa with Diva software (Becton Dickinson). In order to determine the percentage of T regulatory lymphocytes in tumors, the cells were stained with monoclonal antibodies: anti-CD45 PE-Cy7 (BD Biosciences), anti-CD4 FITC (eBioscience), and anti-CD25 PE (eBioscience). Then, cells were fixed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to manufacturer instruction and incubated with anti-FoxP3 monoclonal antibody conjugated with APC (eBioscience). The analysis was carried out using LSR Fortessa with Diva software (Becton Dickinson).

Analysis of MICA expression on patient-derived PCs was performed by gating the CD38⁺CD138⁺ PC population using the antibodies anti-MICA (clone 159227, R&D Systems, Minneapolis, MN, USA), anti-CD38/APC, and anti-CD138/FITC (both from BD Bioscience, San Jose, CA, USA) as previously reported (16 (link)); samples were acquired using a FACSCanto (BD Biosciences, San Jose, CA, USA) and a FACSCalibur (Becton Dickinson). Analysis of NKG2D and DNAM-1 on NK cells from PBMCs or BM aspirates was performed by gating on the CD45⁺CD138⁻CD3⁻CD56⁺ population using the antibodies anti-CD3/allophycocyanin-H7, anti-CD56/PE, anti-CD138/FITC, anti-CD45/PE-Cy7, anti-NKG2D/APC, or anti-DNAM-1/APC (BD Bioscience). In some experiments, anti-CD16/PerCP mAb was also used (BD Bioscience). In the experiments relative to Figure 4 and Figure S2 in Supplementary Material, all the patients-derived samples were acquired using a FACSCanto (BD Biosciences, San Jose, CA, USA). In the experiments relative to Figure 5, PBMCs derived from healthy donors were incubated with medium alone or serum derived from MGUS or MM patients for 16 h. After harvesting, cells were stained with antibodies from BD Bioscience: anti-CD3/PerCP, anti-CD56/APC, and anti-NKG2D/PE; samples were acquired using a FACSCalibur (Becton Dickinson).
Data analysis was performed using the FlowJo 9.3.2 program (TreeStar, Ashland, OR, USA).