Recombinant tpo

E14.5 fetal liver cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 1% of penicillin/streptomycin and cultured for 4 days in the presence of vehicle (PBS), recombinant TPO (50 ng/ml, R&D Systems, Minneapolis, USA), CXCL12 (100 ng/ml, R&D systems), or the combination of both. At day 4, mature MKs were enriched over a bovine serum albumin (BSA) density gradient and lysed for immunoblotting. When used for mRNA analysis, MKs were enriched over a BSA gradient, stained with biotin-conjugated CD41 antibody and further purified using anti-biotin magnetic microbeads (Miltenyi Biotech, San Diego, CA, USA). In order to assess pro-platelet production, MKs were enriched through a BSA density gradient on day 3. Enriched MKs were incubated for an additional 18 h to induce proplatelet formation and the release of nascent platelets. The percentage of MKs forming proplatelets was determined by counting proplatelet-forming and total MKs using an Olympus CKX41 microscope.

The proliferation of Ba/F3 expressing WT or mutants TpoR mutants was assessed by Cell-Titer-Glo luminescent cell viability assay (Promega). Prior to the experiment, cells were washed three times in PBS. 96-wells plate were used to plate the cells at 10,000 cells/well in RPMI +10% FBS supplemented or not by different concentrations of recombinant Tpo (R&D Systems). After 72 hr, ATP-reacting substrate was added following the manufacturer’s instructions and luminescence readings were performed using a Perkin Elmer Victor X luminometer.