Cd45.1 fitc and cd45 2 pe antibodies

Peripheral blood from C3HeB/SJL chimeras was collected by submandibular bleed without anesthesia 6 weeks after BMT, as reported previously.19 (link) Engraftment of donor BM cells was confirmed by double staining of peripheral leukocytes with a cocktail of CD45.1-FITC and CD45.2-PE antibodies (1:500; eBiosciences). In a separate experiment, we collected spleens and blood from SJL and C3HeB mice with or without tail-cuff training (n=8 per group) and processed the samples for flow cytometry analyses, as reported.19 (link) Briefly, mouse spleens were minced in PBS supplemented with 3% FBS. Cells were isolated from the suspension by centrifugation and then resuspended in 3% FBS. Similarly, blood was incubated with 5 mL of ammonium-chloride-potassium lysis buffer for 5 minutes, washed, and resuspended in 3% FBS. Cell viability and cell number were assessed in aliquoted samples (10 μL) using trypan blue staining. Cells (1×10⁶) were incubated with a cocktail of antibodies (eBiosciences) containing Ly6C-PE (1:500), Ly6G-FITC (1:500), CD11c-APC (1:200), and CD11b-PE CY7 (1:1500). For compensation controls, we used IgG beads stained with a single-color antibody. Immunofluorescence was detected using an Accuri C6 flow cytometer (BD Biosciences) and analyzed with the FlowJo software, as described previously.20 (link)^.