Hla dr

To examine the phenotype of CD:UPRT:GFP producing AD-MSC, cells were labelled with MSC Phenotyping Kit consisting of antibodies CD73, CD105, CD14, CD20, CD34, CD45 and HLA-DR (Miltenyi Biotech) according to the manufacturer’s instructions. CD90-PE (Miltenyi Biotech) was used separately for CD90 expression characterization. Expression of the markers were analysed using isotype controls with FACS.

Selected early and senescent passage cells were defrosted from frozen vials in a water bath at 37 °C. The cells were counted to be of at least ${10}^5$ cells per tube. The cell pellet was re-suspended in 200 $\mathrm{\mu }\text{l}$ of blocking buffer (0.5% BSA—bovine serum albumin, 2% FBS in 1× PBS) and incubated for 15 min at room temperature. FACS buffer (0.5% BSA, 0.05% Sodium Azide in 1× PBS) of 200 $\mathrm{\mu }\text{l}$ were added to the suspension and the solution was split into tubes with 50 $\mathrm{\mu }\text{l}$ each. Antibodies against MSC positive markers (Miltenyi Biotec): CD73-PE (Clone AD2), CD90-PerCP-Vio700 (Clone REA897), CD105-FITC (Clone 43A4E1) and negative markers (Viogreen): CD14 (Clone REA599), CD19 (Clone LT19), CD34 (Clone AC136), CD45 (Clone REA747), HLA-DR (Clone REA805) were added and solutions were incubated for 15 min at 4 °C in the dark. FACS buffer of 500 $\mathrm{\mu }\text{l}$ were added to each tube to wash off non-binding antibodies. Tubes of stained and unstained cells were spun down at 400 rcf for 5 min, re-suspended in 500 $\mathrm{\mu }\text{l}$ FACS buffer and the data were acquired by Attune Acoustic Focusing Flow Cytometer (Applied Biosystems). The FlowJo software (version 10.7) (http://www.flowjo.com/solutions/flowjo/downloads) was used for data analysis with debris excluded by gates, and the percentage of cell expressing these surface markers were also recorded.

The MM cell lines RPMI8226 and NCI-H929 were cultured at 37 °C in a 5% CO₂ atmosphere in RPMI1640 (Gibco, Waltham, MA, USA) with 10% fetal bovine serum (FBS, Gibco). G-MDSCs from patients or healthy volunteers were purified using CD33, HLA-DR and CD15 magnetic beads (MiltenyiBiotec, Auburn, CA, USA) according to the manufacturer’s protocol, and the purity of the sorted cells was > 95% using flow cytometry. Female BALB/c nude mice (4–6 weeks) from Beijing Hua Fukang Bioscience Company were housed in a pathogen-free environment. Animal experiments were approved by the Committee on Animal Handling of Huazhong University of Science and Technology (Permit Number: S755).