Monoclonal fitc or pe conjugated anti cd41 antibody

Bone marrow cells were harvested from both femurs of control and TNBS-treated 10-day-old C57BL/6 mice (n=10 each) in Iscove’s modified Dulbecco’s medium supplemented with 1% penicillin/streptomycin, 1% L-glutamine, and 10% FBS (Life Technologies, Carlsbad, CA)(15 (link)). Bone marrow from a few adult mice (9- to 12-week-old) was used for comparison. Red cells were lysed using hypotonic buffer (150 mM NaCl, 10 mM NaHCO₃, 100 mM EDTA) × 10 min at room temperature. Cells were then labeled using monoclonal FITC- or PE-conjugated anti-CD41 antibody (BD Biosciences) and purified using anti-FITC or PE ferromagnetic microbeads (Miltenyi Biotec, San Diego, CA). Mature megakaryocytes were enriched on a discontinuous density gradient of bovine serum albumin (BSA) fraction V (0%, 1.5%, 3.0% in phosphate-buffered saline; BSA from Sigma) (16 (link)). After 30 min, cells that settled at the bottom of the tube by gravity were harvested and enumerated using an automated counter (model TC20, Bio-Rad, Hercules, CA). Cytospin preparations were stained with the Wright-Giemsa stain, and morphometric measurements were made using the software program Image J (17 (link)). Cellular ploidy (after propidium iodide staining) and CD41 expression were measured by flow cytometry (model Accuri C6 plus, BD Biosciences).