An IL-1β driven neutrophil-stromal cell axis fosters a BAFF-rich microenvironment in multiple myeloma
An IL-1β driven neutrophil-stromal cell axis fosters a BAFF-rich microenvironment in multiple myeloma
To remove erythrocytes, unfractionated BM samples were incubated with red blood cell (RBC) lysis buffer (0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA in miliQ water) at a ratio of 1:4 for 5min at room temperature. Fc receptors were blocked with 10% normal human AB serum (Sigma-Aldrich). After blocking, cells were stained for sorting in PBS containing 2% FCS at 4°C with the following antibodies: CD138-FITC (MI15, BioLegend), CD45-PerCP-Cy5.5 (2D1, eBioscience), CD11b-PE (ICRF44, eBioscience), CD14-PECy7 (HCD14, BioLegend), CD163-PE-Dazzle594 (GHI/61, BioLegend), CD3-APCCy7 (HIT3a, BioLegend), CD71-Alexa Fluor 700 (Mem-75, ExBio), CD235a-APC (GA-R2, BD Biosciences), CD16-BV510 (3G8, BioLegend). DAPI (Life Technologies) was used for dead cell exclusion. Granulocytes were sorted as CD45mid/hiCD138-CD235a-CD71-CD3- cells within the granulocytic scatter. Monocytes/macrophages were sorted as CD45mid/hiCD138-CD235a-CD71-CD3- cells within the monocytic scatter. Cells were sorted in DMEM + 10% FCS using a FACSAria III (BD Biosciences) and BD FACSDiva version 5.0 (BD Biosciences). Single cells were encapsulated for cDNA synthesis and barcoded using the Chromium Single Cell 3' Reagent Kit v3 (10X Genomics) Libraries were constructed according to the manufacturer's recommendations (10X Genomics)