Hybri max rbc lysis buffer

Mice were conditioned with CD117-sap immunotoxin and sacrificed after 5 days. Femurs were dissected and crushed using a mortar and pestle. Bone marrow was strained through a 40 μm cell strainer and red blood cells (RBCs) lysed using Hybri-Max RBC lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Bone marrow leukocytes were stained for flow cytometric detection of HSPCs using the following LSK-SLAM markers: lineage cocktail, CD117 (c-Kit), Sca-1, CD48, and CD150. Data were acquired using a BD LSR II (San Jose, CA, USA) or Cytek Aurora (Fremont, CA, USA) flow cytometer. Flow cytometric analysis was performed using FlowJo v10.7.1 (BD, Ashland, OR, USA). Further reagent details are provided in the Supplemental materials.

Peripheral blood was periodically collected from the retro-orbital sinus in 3.8% w/v sodium citrate anticoagulant. CBC analyses were performed using a HemaTrue Veterinary Hematology Analyzer (Heska, Loveland, CO, USA). Remaining whole blood was centrifuged at 2,000 × g for 15 min at 4°C. Plasma was removed and frozen at −80°C until used for downstream analyses. Cell pellets were lysed using Hybri-Max RBC lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), and peripheral blood leukocytes were stained for flow cytometric detection of donor- and host-derived cells using monoclonal antibodies against the following surface markers: CD45.1, CD45.2, CD3, B220 (CD45R), and Gr-1 (Ly-6G/Ly-6C). Data were acquired using a BD LSR II (San Jose, CA, USA) or Cytek Aurora (Fremont, CA, USA) flow cytometer. Flow cytometric analysis was performed using FlowJo v10.7.1 (BD, Ashland, OR, USA). Further reagent details are provided in the Supplemental materials.

Bone marrow derived macrophages (BMDM) were isolated by flushing the tibia and femur from both legs of C57BL/6J mice with DMEM (Gibco). The suspension of bone marrow cells obtained was strained on a 40μM nylon mesh cell retainer (Biolegend). After a wash, the pellet was resuspended and treated 2 minutes with Red Blood Cell (RBC) Lysis Buffer Hybri-Max™ (Sigma-Aldrich) to lyse erythrocytes. After washing and counting, cells were resuspended in DMEM, 10% FBS, 20% L929-conditioned media and seeded to be differentiated into BMDMs over 1 week. Mature BMDM were lifted in cold PBS and cells reseeded at the required density in DMEM, 10% FBS, 5% L929-conditioned media and allowed to rest overnight before stimulation. Human PBMCs were isolated from human blood-derived buffy packs using density gradient centrifugation with Lymphoprep (Stem Cell Technologies) followed by Red Blood Cell lysis using Lysis Buffer Hybri-Max™ (Sigma-Aldrich). PBMCs were resuspended in differentiation media (cRMPI, 10% FBS, 50 ng/mL M-CSF) and monocytes enriched by adherence to plastic and used for subsequent training assays or stimulations.