Hla dr l243 apc cy7

PBMCs were incubated with LIVE/DEAD Fixable AQUA Dead Cells Stain (Thermo Fisher, USA) for 15 min at RT, followed by extracellular staining in Gelatin Veronal Buffer (GVB) (CompTech complement Technology Inc, Tyler, TX) for 30 min at room temperature (RT) to characterize complement staining in monocytes with the following monoclonal antibodies (clones) and fluorochromes: CD14 (M5E2) BV605, CD16 (3G8) PE-Cy7, CD56 (HCD56) BV421, CD66b (G10F5) Pacific Blue from Biolegend, HLA-DR (L243) APC-Cy7, C3 (1H8) PE and CD59 (p282(H19)) BV711 from BD bioscience, CD2 (RPA-2.10) eFluor 450, CD3 (UCHT1) eFluor 450, CD19 (SJ25C1) eFluor 450 and CD20 (2H7) eFluor 450 from Life Technology and, finally, C1q (polyclonal) FITC from Dako. Data were acquired on a BD Fortessa flow cytometer (BD Biosciences). All compensation and gating analyses were performed using FlowJo 10.5.3 (TreeStar, Ashland, OR, USA).

Flow cytometry was used for phenotype analyses of cultured and xenografted monocytic AML cell lines and primary AML cells. For all flow cytometry analyses, a minimum of 30,000 gated live cells were analyzed on a four-laser BD LSRFortessa (405, 488, 532, and 640 nm; BD Biosciences). Data analysis was performed using FACSDiva software version 8.0.1 (BD Biosciences).
The following anti-human monoclonal antibodies were purchased from BD Biosciences: CD33 (P67.6)-PE-Cy7, CD34 (8G12)-PE, CD11b (ICRF44)-PE, HLA-DR (L243)-APC-Cy7, and CD45 (HI30)-FITC. The secondary antibodies, rat anti-mouse IgG1 (A85-1)-BV421 and rat anti-mouse CD45 (30-F11)-AF700 were also obtained from BD Biosciences. CD14 (TüK4)-Qdot655, goat anti-rabbit IgG-PE-Cy5.5, DAPI, and Live/Dead Fixable Yellow Dead Cell stain were purchased from Thermo Fisher Scientific. Primary anti-H₂R (LS-A1176) antibody was obtained from LifeSpan Biosciences, Inc. (Seattle, WA, USA) and primary anti-FPR2 (GM1D6) antibody was obtained from Santa Cruz (Heidelberg, Germany). Anti-flavocytochrome b₅₅₈ (NOX2; 7D5)-FITC was purchased from MBL International Corporation (Woburn, MA, USA) and anti-FPR1 (#350418)-APC was from R&D Systems (Minneapolis, MN, USA).