Purified rat anti mouse cd16 cd32 fc block

Cells at D3 CMP stage were treated with 20 nM RIST4721 (or DMSO) and differentiated into mature neutrophils as described above while replenishing the drug and DMSO during culture expansion or media changes. Cell surface marker analysis using fluorescent immunolabeling and imaging flow cytometry was then performed as previously described [42 (link)]. Briefly, the cells were harvested at 1 × 10⁶ per sample through centrifugation at 250× g for 5 min. The pellets were then washed in PBS and centrifuged again. The cells were resuspended in PBS with 2% FBS at 100 μL per sample, and Fc Block (Purified Rat Anti-Mouse CD16/CD32; BD Biosciences) was added into each sample at 50 μg/mL followed by incubation on ice for 15 min. The cells were then stained with PE-conjugated anti-Cd11b, FITC-conjugated anti-Ly6G, or corresponding isotypes (4 μg/mL for each antibody; BD Biosciences) for 45 min on ice while protected from light. Finally, the samples were washed in 400 μL of PBS with 2% FBS, centrifuged at 1400× g for 5 min and resuspended in 35 μL of fresh PBS with 2% FBS for processing with FlowSight imaging flow cytometer (Luminex, Austin, TX, USA).

Blood was collected into heparin-coated tubes by cardiac puncture immediately following CO₂ euthanization. Spleen, BM, cervical LN, mesenteric LN, and PP were harvested and dissociated into single cell suspensions in Dulbecco’s 1× PBS with 2% fetal bovine serum, (Stem Cell Technologies), plus 2 mM EDTA, (Ambion). Red blood cell (RBC) lysis was performed using 1× RBC lysis buffer (eBioscience) or ACK Lysing Buffer (Life Technologies). All staining was performed using LIVE/DEAD Fixable Aqua or Blue Stain (Invitrogen; 15 minutes at room temperature) and Fc block (Purified Rat Anti-Mouse CD16/CD32; BD Pharmingen, 5 minutes at room temperature). Surface staining was completed with the indicated directly conjugated antibodies for 30 minutes on ice. Foxp3 and Bcl-6 staining (eBioscience) were executed according to the manufacturers instructions. Antibodies to CD3, CD4, CD8, CD11b, CD11c, CD25, CD45, B220, CD62L, CD44, CD69, CD127, PD1 (RPMI-30), NKp46, Ly6C, and Ly6G were purchased from BioLegend (San Diego, CA). Antibodies to CD115, Bcl-6 and Foxp3 were purchased from eBioscience (San Diego, CA). Antibodies to CD19, CD49b, CD138, F4/80, CXCR5, ICOS, were purchased from BD Biosciences (San Jose CA). Samples were fixed with 1× Stabilizing fixative and collected using FACSCanto or Fortessa Flow Cytometers (BD Biosciences, San Jose, CA). Data were analyzed by FlowJo Software (Tree Star).

Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated specific antibodies against mouse TER119 (clone TER-119) and mouse CD71 (clone C2F2), and purified rat anti-mouse CD16/CD32 (Fc Block) were purchased from BD Biosciences. Hemin was from Sigma-Aldrich. Recombinant human erythropoietin (EPO) was obtained from R&D systems. Cell counting kit-8 (CCK-8) was from Dojindo Molecular Technologies, Inc. Alexa Fluor 647-conjugated cholera toxin subunit B (CTB) was from Life Technologies. All other chemicals were of the highest grade available from commercial sources.