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Comparison of gene expression profiles of p45-/- primary megakaryocytes and WT primary megakaryocytes

ABSTRACT: Whole livers were collected from mouse fetuses at embryonic day 13.5 (E13.5), and single-cell suspensions were prepared by successive passage through 25-gauge needles. Fetal liver cells were maintained in RPMI1640 (Wako, Osaka, Japan) supplemented with 20% charcoal-stripped fetal bovine serum (FBS), 100 U/ml penicillin, 100µg/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO) (generously provided by Kyowa Hakko Kirin Co. Ltd.). Megakaryocytes were harvested for RNA purification from a day-3 culture. CD41+ cells were enriched using biotinylated anti-CD41 antibody (Serotec; clone MWReg30) and streptavidin-coupled Dynabeads (Dynal Biotech ASA) from a culture of E13.5 fetal liver cells of WT and p45–/– mice. Total RNA was purified from the sorted CD41+ cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE35645 | GEO | 2013/08/07

SECONDARY ACCESSION(S): PRJNA152501

REPOSITORIES: GEO

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Project description:Normal donors BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Essential Throbocytemia BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Keywords: other