Cd45 fitc clone hi30

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.

To determine cellular origin of circulating B cell activating factor (BAFF) + MVs, flow cytometric analysis was performed on selected PROFLOW cohort samples with an Apogee A60 cytometer (Apogee Flow Systems, Hemel Hempstead, UK). Size-calibrated silica beads (ApogeeMix, Apogee Flow Systems) were used to establish MV gate (Supplemental Fig. S1). Fluorescence gates were created based on Ab isotype controls: PE mouse IgG1κ, FITC mouse IgG1κ, and Alexa Fluor 488 mouse IgG1κ (Supplemental Fig. S2), as well as Abs in DBPS. All Abs were centrifuged 20,000 g for 30 min in 4°C prior application, to remove Ab aggregates. Patient plasma was diluted 1:200 with DPBS, and 200 μL was stained for 30 min in RT with 2 μL of Abs against cellular origin markers and BAFF and directly analyzed. The gating strategy is presented in Supplemental Fig. S3A. CD42b PE and FITC clone HIP1, CD31 PE clone WM59, CD45 FITC clone HI30, CD16 PE and FITC clone 3G8, CD62E PE clone 68-5H11, CD36 PE clone CB38, and CD144 FITC clone 55-7H1 were purchased from BD Biosciences. CD235a FITC clone KC16 was purchased from Beckman Coulter. BAFF Alexa Fluor 488 was purchased from Bio-Techne. BAFF PE clone 1D6 was purchased from Thermo Fisher Scientific.