Journal:

Article Title: Targeted Disruption of the Murine fps/fes Proto-Oncogene Reveals that Fps/Fes Kinase Activity Is Dispensable for Hematopoiesis

doi:

Figure Lengend Snippet: BM hematopoietic progenitor cell colony assays. (A) BM cells were grown in the presence of a cocktail of cytokines, consisting of IL-3 (5 ng/ml), IL-6 (10 ng/ml), SF (50 ng/ml), and Epo (1 U/ml). (B) BM cells were grown in the presence of GM-CSF (5 ng/ml) and Epo (1 U/ml). Bars in panels A and B: white, total number of colonies; hatched, CFU-GM and CFU-M colonies combined; stippled, BFU-E colonies; black, CFU-GEMM colonies. Colony counts were made on day 8 postplating. In panel C, CFU-E colonies were grown from BM cells under the following conditions: the cytokine cocktail (white bars), GM-CSF and Epo (hatched bars), and Epo alone (black bars). CFU-E colony counts were made 2 days postplating. The sample sizes were six mice per genotype for each panel. The height of each bar represents the average number of colonies for the six mice analyzed, and the error bars represent SD. In panel D, 20 CFU-GM colonies were randomly picked from 1.2-ml methylcellulose cultures which had been seeded with 50,000 nucleated BM cells. Colonies were picked 9 days postplating, and cells were pooled and counted. The height of each bar represents the average number of cells per CFU-GM colony, and error bars indicate SD. The sample sizes were four mice of each genotype.

Article Snippet: Briefly, 50,000 unfractionated BM cells were cultured in 1.2 ml of semisolid medium consisting of 1% methylcellulose (Fluka), 10% fetal bovine serum (FBS), 2% bovine serum albumin (BSA), 200 μg of human plasma transferrin (iron saturated; (Calbiochem) per ml, 50 μM α-monothioglycerol (Sigma), and either (i) a cytokine cocktail consisting of recombinant human Epo (1 U/ml; R&D Systems), recombinant murine IL-3 (5 ng/ml; (R&D Systems), recombinant murine Steel factor (SF) (50 ng/ml; R&D Systems), and recombinant murine IL-6 (10 ng/ml; R&D Systems); (ii) recombinant murine GM-CSF (5 ng/ml; R&D Systems) and recombinant human Epo (1 U/ml); or (iii) recombinant human Epo (1 U/ml) alone.

Techniques:

Journal:

Article Title: Targeted Disruption of the Murine fps/fes Proto-Oncogene Reveals that Fps/Fes Kinase Activity Is Dispensable for Hematopoiesis

doi:

Figure Lengend Snippet: Signaling in cultured fpsK588R homozygous mutant BMM. BMM from wild-type (+/+) or homozygous mutant (−/−) mice were starved for 48 h in 0.5% FBS prior to stimulation with either GM-CSF (30 ng/ml) or IL-6 (30 ng/ml). Cells were exposed to each cytokine for 15 min at 37°C, scraped in 2× SDS sample buffer, run out on an SDS–7.5% polyacrylamide gel, transferred to Immobilon-P membrane, and then probed successively with the indicated antibodies: (A) anti-pStat3 (top) followed by anti-Stat3 (bottom); or (B) anti-pStat5A/B (top) followed by anti-Stat5A (bottom). Stat5B (90 to 92 kDa) is constitutively phosphorylated in these BMM cultures, even under starvation conditions, whereas Stat5A (95 kDa) is phosphorylated only following GM-CSF stimulation. (C) BMM were starved for 48 h in 0.5% FBS prior to exposure to various doses of LPS for 30 min at 37°C. Whole-cell lysates were run out on SDS–11% polyacrylamide gels, transferred to Immobilon-P membrane, and then probed with anti-Erk antibody (bottom) followed by anti-pErk antibody (top).

Article Snippet: Briefly, 50,000 unfractionated BM cells were cultured in 1.2 ml of semisolid medium consisting of 1% methylcellulose (Fluka), 10% fetal bovine serum (FBS), 2% bovine serum albumin (BSA), 200 μg of human plasma transferrin (iron saturated; (Calbiochem) per ml, 50 μM α-monothioglycerol (Sigma), and either (i) a cytokine cocktail consisting of recombinant human Epo (1 U/ml; R&D Systems), recombinant murine IL-3 (5 ng/ml; (R&D Systems), recombinant murine Steel factor (SF) (50 ng/ml; R&D Systems), and recombinant murine IL-6 (10 ng/ml; R&D Systems); (ii) recombinant murine GM-CSF (5 ng/ml; R&D Systems) and recombinant human Epo (1 U/ml); or (iii) recombinant human Epo (1 U/ml) alone.

Techniques: Cell Culture, Mutagenesis, Membrane