Journal: PLoS ONE

Article Title: Membrane-Bound Steel Factor Maintains a High Local Concentration for Mouse Primordial Germ Cell Motility, and Defines the Region of Their Migration

doi: 10.1371/journal.pone.0025984

Figure Lengend Snippet: (A) There was no significant change in PGC numbers at E7.5 in Steel d/d embryos compared to their littermates. “n” indicates the number of embryos used for quantitation. (B) PGC number after 24 hours in vitro culture in medium with or without soluble recombinant Steel factor on different feeder layer cells. Y axis represents the ratio of PGC number 24 hours after plating versus 3 hours after plating. ΔMEF: primary MEF from Steel-null embryos. M220: stromal cell line express only membrane-bound Steel factor. ** = p<0.01. (C) PGC number reduced significantly in E8.0 Steel d/d embryos compared to their littermates. “n” indicates the number of embryos used for quantitation. * = p<0.05.

Article Snippet: For the Steel factor addition assay, 200 ng/ml soluble mouse recombinant Steel factor (R&D Systems) was added into the embryo culture medium immediately before time-lapse analysis was started.

Techniques: Quantitation Assay, In Vitro, Recombinant, Membrane

Journal: PLoS ONE

Article Title: Membrane-Bound Steel Factor Maintains a High Local Concentration for Mouse Primordial Germ Cell Motility, and Defines the Region of Their Migration

doi: 10.1371/journal.pone.0025984

Figure Lengend Snippet: (Column A, B) Frames at t = 0 and t = 6 hours respectively from movies of E7.5 Steel d/d embryos with or without addition of 200 ng/ml soluble recombinant Steel factor. (Column C) Tracks were made from PGCs in the allantois (white boxes) that remained in the plane of the confocal image throughout the movies. The white line indicates the boundary between the extraembryonic tissues (EEM), and the posterior end of the embryo (PEM). Scale bars in (A–C): 100 µm. (D) The maximum velocity, average velocity, and displacement of PGCs in E7.5 Steel d/d embryos were significantly increased by adding of 200 ng/ml soluble recombinant Steel factor into culture medium for 6 hours. “n” indicates the number of PGCs used for quantitation. Units on the “Y” axis vary based upon parameter, and are indicated below the bar charts. ** = p<0.01. (E) The maximum velocity, average velocity, and displacement of PGCs after 24 hours in vitro culture with increasing concentration of soluble recombinant Steel factor on Steel-null MEFs (ΔMEF). Units on the “Y” axis vary based upon parameter, and are indicated below the bar charts. * = p<0.05.

Article Snippet: For the Steel factor addition assay, 200 ng/ml soluble mouse recombinant Steel factor (R&D Systems) was added into the embryo culture medium immediately before time-lapse analysis was started.

Techniques: Recombinant, Quantitation Assay, In Vitro, Concentration Assay

Journal: PLoS ONE

Article Title: Membrane-Bound Steel Factor Maintains a High Local Concentration for Mouse Primordial Germ Cell Motility, and Defines the Region of Their Migration

doi: 10.1371/journal.pone.0025984

Figure Lengend Snippet: Directions of individual PGC migration in Steel d/d embryos (A) or wild type embryos (B) with or without addition of 200 ng/ml soluble recombinant Steel factor. The boundary between the extraembryonic tissues (EEM), and the posterior end of the embryo (PEM), is marked by a line. Column I, II, and III are representative images from 3 different embryos of the same genotype labeled on the left. (C) The maximum velocity, average velocity, and displacement of PGCs in E7.5 wild type embryos with or without addition of 200 ng/ml soluble recombinant Steel factor into culture medium for 6 hours. “n” indicates the number of PGCs used for quantitation. Units on the “Y” axis vary based upon parameter, and are indicated below the bar charts. ** = p<0.01.

Article Snippet: For the Steel factor addition assay, 200 ng/ml soluble mouse recombinant Steel factor (R&D Systems) was added into the embryo culture medium immediately before time-lapse analysis was started.

Techniques: Migration, Recombinant, Labeling, Quantitation Assay

Journal: PLoS ONE

Article Title: Membrane-Bound Steel Factor Maintains a High Local Concentration for Mouse Primordial Germ Cell Motility, and Defines the Region of Their Migration

doi: 10.1371/journal.pone.0025984

Figure Lengend Snippet: (Column A-C) Frames at t = 0, t = 6 and t = 12 hours respectively from movies of E11.0 wild type embryo slices with or without addition of 200 ng/ml soluble recombinant Steel factor. (D) The relative expression level of membrane-bound and soluble Steel factor mRNA in E10.5 genital ridges, E11.5 genital ridges and E11.5 midline mysenchyme as determined by real-time RT-PCR. *p<0.05, ** p <0.01. (E and F) E-cadherin expression in PGCs without (E) or with (F) addition of Steel factor. Upper panels show E-cadherin (red) staining. The lower panels show the merged images with PGC marker Stella-GFP (green). No difference in expression of E-cadherin was observed between groups.

Article Snippet: For the Steel factor addition assay, 200 ng/ml soluble mouse recombinant Steel factor (R&D Systems) was added into the embryo culture medium immediately before time-lapse analysis was started.

Techniques: Recombinant, Expressing, Membrane, Quantitative RT-PCR, Staining, Marker

Journal: Frontiers in Immunology

Article Title: GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

doi: 10.3389/fimmu.2017.00147

Figure Lengend Snippet: Granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates c-kit and stem cell factor (SCF) expression by BM-derived DCs (BMdDCs) . (A–C) Effect of GM-CSF and cell density on c-kit expression by BMdDCs. BMdDCs were plated in 24-well plates and cultured for 2 days in complete Opti-MEM medium in four different conditions, that is at either 2 × 10 5 or 1.2 × 10 5 cell/well, and either with or without (w/o) GM-CSF at 20 ng/ml, as indicated. Cells were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry. (A) Typical flow cytometric profiles, showing CD40 and MHCII expression by BMdDCs. Numbers represent percentages of cells in the indicated regions. (B) Typical histograms showing c-kit expression by MHCII int CD40 int and MHCII hi CD40 hi BMdDCs, gated as in (A) . Solid lines represent c-kit staining profiles, dashed lines represent isotype control mAb. Numbers indicate c-kit median fluorescence intensity (MFI) values. (C) Summary of c-kit expression results obtained from MHCII hi CD40 hi BMdDCs, gated as in (A) . c-kit MFI from individual samples and average values (bar). (D,E) Effect of GM-CSF on SCF expression by BMdDCs. (D) Cell lysates were prepared from day 0 BMdDCs and BMdDCs cultured for 2 days in 24-well plates at 1.2 × 10 5 cell/well in complete Opti-MEM medium with or w/o GM-CSF at 20 ng/ml, as indicated. SCF protein expression was analyzed by ELISA, testing 25 µg of cell lysate in 100 µl/well. Data are expressed as picograms per milliliter. Individual results from three experiments and average values (bar) are shown. (E) Day 0 and day 2 BMdDCs cultured in 24-well plates at 1.2 × 10 5 cell/well with or w/o GM-CSF at 20 ng/ml were analyzed by Real-Time PCR in triplicates. SCF mRNA expression was calculated relative to hprt1 in arbitrary units. For each experiment, day 2 c-kit/hprt1 levels were normalized with day 0. In (A,B) representative data of N = 4 experiments, in (C) N = 4 experiments, in (D) N = 3 experiments, in (E) mean ± SD of four experiments (* P ≤ 0.05; ** P ≤ 0.01).

Article Snippet: BMdDC culture supernatants (100 μl/well) and BMdDC lysates (25 μg of cell lysate/well) were tested by mouse SCF ELISA kit (Boster Immunoleader, Pleasanton, CA, USA).

Techniques: Expressing, Derivative Assay, Cell Culture, Staining, Bioprocessing, Flow Cytometry, Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction