Pe mouse anti human cd33

Antibodies (Abs) used for flow cytometry, PE mouse antihuman CD33 (Cat#: 555,450) and APC mouse antihuman CD45 (Cat#: 555,485) were purchased from BD Biosciences, PerCP/Cy5.5 antihuman CD19 (Cat#: 302,229) was purchased from Biolegend. Cytarabine (Cat#: BD8499), imatinib (Cat#: BD42606), and ibrutinib (Cat#:BD254580) were purchased from Bide Pharmatech (Shanghai, China). Epirubicin (Cat#: 56,390-09-1) was purchased from Shandong New Time Pharmaceutical Co., Ltd., Company (Shandong, China). Vincristine (Cat#: MB1298) was purchased from Melone Pharma (Dalian, China). Cladribine (Cat#:CSN10004) was purchased from CSNpharma (Shanghai, China).

Cells were analyzed using Flow Cytometer (Cytomic FC500, Beckman). Tissues were trim into 1-2mm³ tissue block and put into burnisher. Then cell suspension was collected and centrifugated. To blood specimen, heparin sodium and PBS/Hanks was added into blood of individuals. Ficol was used to separate lymphocyte. Serum free medium containing 1% BSA was added into cell suspension, and then incubated on ice for 10 minutes. PE-Cy Mouse Anti-Human CD11b, FITC-Mouse Anti-Human LIN, APC Mouse Anti-Human HLA-DR, PE Mouse Anti-Human CD33 (BD Biosciences) were added to cell suspension and incubated on ice for 30 minutes. Cells were washed and resuspended in 500 mL buffer and analyzed.

Peripheral blood mononuclear cells (PBMCs) from blood samples were separated by Ficoll-Hypaque density gradient centrifugation. And MDSCs were sorted from PBMCs by using CD33 labeled magnetically selection monoclonal antibodies (Miltenyi Biotech, Germany). PE mouse anti-human CD33, PE-Cy7 mouse anti-human CD11b, APC mouse anti-human HLA-DR and FITC mouse anti-human LIN (BD Biosciences, USA) were applied to examine the proportion of MDSCs by Flow Cytometry (Cytomic FC500, Beckman). WinMDI was used to analyze samples.