Platelets are anuclear and lack nuclear DNA. They contain a rough endoplasmic reticulum and polyribosomes, and retain the ability to synthesize proteins from cytoplasmic mRNA. The platelets play critical roles in normal hemostatic processes and pathologic conditions such as thrombosis, vascular remodeling, inflammation, and wound repair.
Platelets are collected by apheresis. After addition of EDTA (2 mM), apheresis-derived platelets from a single donor are centrifuged at 140g for 15 minutes at 25°C. To minimize leukocyte contamination, only the upper 9/10 of the platelet-rich plasma (PRP) is passed through a BioGel A50M column (1000 mL total volume) equilibrated with HBMT (HEPES-buffered modified Tyrodes buffer (HEPES (10 mM) pH 7.4, NaCl (150 mM), KCl (2.5 mM), NaH2PO4 (0.3 mM), NaHCO3 (12 mM), bovine serum albumin [0.2% BSA], glucose (0.1%), EDTA (2 mM)). Gel-filtered platelets (GFPs) are subsequently filtered through a nonwetting nylon filament filter (5-μm) at 25°C and harvested by centrifugation at 1500g for 10 minutes at 25°C. Platelets are gently and thoroughly resuspended in 10 mL HBMT buffer and incubated with 120 μL murine monoclonal anti-CD45 antibody conjugated to magnetic microbeads on a rotating platform for 45 minutes at 25°C. Magnetic separation columns are used to capture CD45+ cells (leukocyte fraction).
EMBRYONIC DEVELOPMENT & STEM CELL COMPENDIUM
