Cd33 pe cy7 clone p67.6

Multiparameter analyses of stained cell suspensions were performed on FACS CANTO II (BD Bioscience) and analyzed with FlowJo v10.4 software (Treestar).
To quantify the level of TNAP in BMSCs after co-culture with AML or ALL cell lines, primary AML cells, or healthy CD34⁺ progenitors, the adherent layer was subjected to trypsinization after washing with PBS to remove non-adherent cells. Cells were stained with TNAP-PE (clone W8B2; Biolegend) and with a fluorochrome-conjugated mAb specific to exclude residual leukemia cells. Specifically, CD33-PE-Cy7 (clone P67.6; BD Bioscience) was used to gate AML cells, CD19-PE-Cy7 (clone J3-119; Beckman Coulter) was used to gate B-ALL cells and CD34-FITC (clone 581; BD Pharmingen) was used to gate CD34⁺ progenitors.
The following antibodies were used for the analysis of BMSCs: CD90-PE (clone 5E10; eBioscience), CD73-PE (clone AD2; BD Pharmingen), CD105-PE (clone SN6; eBioscience), CD146-PE (clone P1H12; BD Pharmingen), CD45-FITC (clone HI30, BD Pharmingen), and CD34-FITC.
Median fluorescence intensities were calculated in co-cultured BMSCs in comparison to control with FlowJo software.

Freshly prepared PBMC were treated with FcR Blocking Reagent (130-111-568, Miltenyi Biotec) according to the manufacturer’s protocol and stained with fixable viability dye 700 (BD Biosciences) followed by the incubation with monoclonal antibodies (mAbs) for 30 min at 4°C. The following fluorescently labeled mAbs were used for the surface staining: CD66b-PerCPCy5.5 (clone G10F5), CD14-APCCy7 (clone MФP9), HLA-DR-V500 (clone G46-6), lineage cocktail (LIN) (CD3/19/20/56)-APC, CD33-PE-Cy7 (clone P67.6), CD39-FITC (clone TU66), PD-L1-BV421 (clone MIH1, all from BD Biosciences) and CD73-BV605 (clone AD2, Biolegend). Intracellular ROS and NO were detected using hROS Detection Kit (Cell Technology) and diaminofluorescein-FM diacetate (Cayman Chemical) according to the manufacturer’s instructions. Acquisition was performed by 10-color flow cytometry using BD FACSLyric with FACSuite software (BD Biosciences). FlowJo V 10 software (BD Biosciences) was used to analyze at least 10⁶ events. Positive surface markers were gated according to the fluorescence minus one (FMO) control.