Journal: Frontiers in Immunology

Article Title: The Culture Dish Surface Influences the Phenotype and Cytokine Production of Human Monocyte-Derived Dendritic Cells

doi: 10.3389/fimmu.2019.02352

Figure Lengend Snippet: Influence of the culture dish on the phenotype of differentially stimulated moDC. The phenotype was analyzed by flow cytometry using the indicated surface molecules. % positive cells or median fluorescence intensity (MFI) are shown. Color code: Control (DMSO)—green, immunogenic DC populations (LPS & Jonuleit cytokine cocktail)—red, tolerogenic DC populations (Dexamethasone with vitamin D3 & IL-10)—blue. Squares (left) : standard culture dish; circles (right) : non-adherent culture dish. The median is marked with a line. Significance values are given in grades * P < 0.05, ** P < 0.01, and *** P < 0.001. Black significance grades are the result of a collective 2 way ANOVA testing for all treatments, red grades are from separate tests for the iDC/tolDC and immunogen subgroups ( n = 8).

Article Snippet: LPS (100 ng/ml; Sigma-Aldrich, Taufkirchen, Germany) and the Jonuleit cytokine cocktail [10 ng/ml of IL-1β, 10 ng/ml of TNF, 1,000 U/ml of IL-6; all ImmunoTools, Friesoythe, Germany, and 1 μg/ml of prostaglandin E2 (PGE 2 ); SigmaAldrich, Taufkirchen, Germany] were added 24 h before harvesting, respectively.

Techniques: Flow Cytometry, Fluorescence, Control

Journal: Frontiers in Immunology

Article Title: The Culture Dish Surface Influences the Phenotype and Cytokine Production of Human Monocyte-Derived Dendritic Cells

doi: 10.3389/fimmu.2019.02352

Figure Lengend Snippet: Influence of the culture dish on the cytokine production of differentially stimulated moDC. Cytokines were measured in cell free culture supernatants of the generated moDC populations. Color code: Control (DMSO)—green, immunogenic DC populations (LPS and Jonuleit cytokine cocktail, respectively)—red, tolerogenic DC populations (Dexamethasone with vitamin D3 and IL-10, respectively)—blue. Circles (left) : standard culture dish; squares (right) : non-adherent culture dish. The median is marked with a line. Significance values are given in grades * P < 0.05, ** P < 0.01, and *** P < 0.001. Black significance grades are the result of a collective 2 way ANOVA testing for all treatments, red grades are from separate tests for the iDC/tolDC and immunogen subgroups ( n = 6).

Article Snippet: LPS (100 ng/ml; Sigma-Aldrich, Taufkirchen, Germany) and the Jonuleit cytokine cocktail [10 ng/ml of IL-1β, 10 ng/ml of TNF, 1,000 U/ml of IL-6; all ImmunoTools, Friesoythe, Germany, and 1 μg/ml of prostaglandin E2 (PGE 2 ); SigmaAldrich, Taufkirchen, Germany] were added 24 h before harvesting, respectively.

Techniques: Generated, Control

Journal: Frontiers in Immunology

Article Title: The Culture Dish Surface Influences the Phenotype and Cytokine Production of Human Monocyte-Derived Dendritic Cells

doi: 10.3389/fimmu.2019.02352

Figure Lengend Snippet: Antigen-specific T cell induction is not influenced by the culture dish conditions of the moDC. Autologous PBMC depleted of monocytes were co-cultured with PPD-loaded DC generated on the indicated surface and matured with indicated stimuli for 7 days, and antigen-specific IFN-γ secretion by CD4+ and CD8+ cells was analyzed by flow cytometry with LPS-DC as stimulators (± PPD). Delta LPS: moDC cultured on a conventional culture dish, stimulated with LPS; Hydrocell LPS: moDC cultured on a non-adherent culture dish, stimulated with LPS; Delta Jonuleit: moDC cultured on a conventional culture dish, stimulated with Jonuleit cytokine cocktail (TNF, IL-6, IL-1β, and PGE 2 ); Hydrocell Jonuleit: moDC cultured on a non-adherent culture dish, stimulated with Jonuleit cytokine cocktail. Each color-coded symbol represents results from one individual ( n = 9).

Article Snippet: LPS (100 ng/ml; Sigma-Aldrich, Taufkirchen, Germany) and the Jonuleit cytokine cocktail [10 ng/ml of IL-1β, 10 ng/ml of TNF, 1,000 U/ml of IL-6; all ImmunoTools, Friesoythe, Germany, and 1 μg/ml of prostaglandin E2 (PGE 2 ); SigmaAldrich, Taufkirchen, Germany] were added 24 h before harvesting, respectively.

Techniques: Cell Culture, Generated, Flow Cytometry

Journal: Biology of Reproduction

Article Title: Loss of Raptor induces Sertoli cells into an undifferentiated state in mice

doi: 10.1093/biolre/ioac104

Figure Lengend Snippet: Female steroidogenic activity and estradiol levels were elevated in the SC Raptor KO gonads. (A) qRT-PCR shows the expression profiles of the enzymes involved in male and female steroidogenesis in control and mutant gonads from 5 to 60 dpp. Age-match WT ovaries were included for comparison. (B) Raised estradiol levels in the adult mutant gonads were revealed by ELISA. The data are present as pg per mg protein. Adult WT ovaries were also included for comparison. Note that all four control testes assayed had levels below the detection limit and thus were defined as zero, whereas two out of four mutant testes had measurable estradiol. (C) Reduced testosterone levels in the adult mutant gonads. The data are present as ng per mg protein. (D and E) Measurement of 17-OH-progesterone (D) and androstenedione (E) by UPLC-MS/MS. Both data are present as pg per mg tissue mass. (F) Co-immunofluorescence of α-SMA and ZP3, an oocyte-specific zona pellucida protein, in control and mutant gonads from 5 to 60 dpp and adult WT ovaries. * , P < 0.05; * * , P < 0.01; * * * , P < 0.001. Scale bar = 100 μm.

Article Snippet: After centrifuging at 12 000 rpm at 4°C for 15 min, the supernatant was collected for testosterone and estradiol measurement by using the enzyme-linked immunosorbent assay (ELISA) kits (testosterone, E-EL-0072c, detection level: 0.10–20 ng/ml; estradiol, E-EL-0150c, 9.37–600 pg/ml; both from Elabscience, Wuhan, China).

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Control, Mutagenesis, Comparison, Enzyme-linked Immunosorbent Assay, Tandem Mass Spectroscopy, Immunofluorescence

Journal: NPJ Regenerative Medicine

Article Title: Machine learning-based classification of dual fluorescence signals reveals muscle stem cell fate transitions in response to regenerative niche factors

doi: 10.1038/s41536-023-00277-4

Figure Lengend Snippet: a Schematic depicting the time course of MuSC treatment with PGE2. b Representative immunofluorescence images of MuSCs after 7 days of culture, treated with PGE2 or vehicle. c Quantification of cell number reveals increased proliferation in PGE2-treated MuSCs by day 3 in culture. d Quantification of myogenic factor expression 7 days after PGE2 treatment reveals expansion of the stem cell pool. e Schematic depicting the time course of MuSC treatment with OSM. f Representative immunofluorescence images of MuSCs after 7 days of culture, treated with OSM or vehicle. g Quantification of cell number reveals decreased proliferation in OSM-treated MuSCs at day 7 in culture. h Quantification of myogenic factor expression 7 days after OSM treatment reveals a bias toward stemness maintenance and decreased commitment in OSM-treated MuSCs. In ( d and h ), cells that were Pax7+ and MyoG− were considered part of the “stem” population, cells that were MyoD+, Pax7−, and MyoG− were considered part of the “early commitment” population, and cells that were Pax7− and MyoG+ were considered part of the “terminal commitment” population. * p < 0.05, ** p < 0.01, two-tailed Student’s t test. Data in bar graphs are represented as mean ± standard deviation. In ( d and h ), the color of the stars denoted the pairwise comparison performed (stem, early commitment, or terminal commitment). In ( c and d ), n = 7 independent replicates for controls and 4 independent replicates for PGE2 treatment. In ( g and h ), n = 8 independent replicates for controls and 3 independent replicates for OSM treatment. In ( b and f ), scale bars represent 50 µm.

Article Snippet: PGE2 (10 ng/mL, Cayman Chemical, Ann Arbor, MI) or OSM (100 ng/mL, R&D Systems, Minneapolis, MN) was then administered, and time lapse imaging was started.

Techniques: Immunofluorescence, Expressing, Two Tailed Test, Standard Deviation, Comparison

Journal: NPJ Regenerative Medicine

Article Title: Machine learning-based classification of dual fluorescence signals reveals muscle stem cell fate transitions in response to regenerative niche factors

doi: 10.1038/s41536-023-00277-4

Figure Lengend Snippet: a Schematic depicting the relative timing of four transcription factors that determine MuSC fate. b Schematic depicting the workflow of binary reporter time lapse experiments to determine how PGE2 and OSM regulate MuSC fate decisions. c Graphic depicting the Dual-FLIT approach and how the binary fluorescent signals enable classification of cell fate decisions. d Representative fluorescence and brightfield micrograph of a single MuSC clone expressing the eGFP Pax7-reporter and the mCherry MyoG-reporter. Scale bar: 100 µm. e Single MuSCs seeded in hydrogel microwells were imaged over time and tracked using the Baxter Algorithm. f Representative lineage trees from vehicle, PGE2, or OSM treated MuSCs from d3 to d6 (72 h) were generated by adding fluorescent reporter and machine learning classification modules to the Baxter Algorithm workflow. Division events were classified as symmetric self-renewing (“SR”), asymmetric (“A”), or symmetric committed (“SC”).

Article Snippet: PGE2 (10 ng/mL, Cayman Chemical, Ann Arbor, MI) or OSM (100 ng/mL, R&D Systems, Minneapolis, MN) was then administered, and time lapse imaging was started.

Techniques: Fluorescence, Expressing, Generated

Journal: NPJ Regenerative Medicine

Article Title: Machine learning-based classification of dual fluorescence signals reveals muscle stem cell fate transitions in response to regenerative niche factors

doi: 10.1038/s41536-023-00277-4

Figure Lengend Snippet: The Dual-FLIT approach enables tracking of individual MuSCs and their progeny through numerous rounds of cell division as shown in color-code by generation. Dead cells each in each condition are shown in black. Treatment with PGE2 accelerates cell division, with some microwells having progeny in the 8th generation after 3 days. In contrast, OSM slows down proliferation, acting as a pro-quiescence factor. At least 18 independent clones were tracked for each condition from at least 2 independent experimental replicates.

Article Snippet: PGE2 (10 ng/mL, Cayman Chemical, Ann Arbor, MI) or OSM (100 ng/mL, R&D Systems, Minneapolis, MN) was then administered, and time lapse imaging was started.

Techniques: Clone Assay

Journal: NPJ Regenerative Medicine

Article Title: Machine learning-based classification of dual fluorescence signals reveals muscle stem cell fate transitions in response to regenerative niche factors

doi: 10.1038/s41536-023-00277-4

Figure Lengend Snippet: a Cumulative division counts and classifications for vehicle, PGE2, and OSM treated MuSCs, normalized to represent the same number of starting clones per condition. b Classification of division types broken down by generation reveals temporal dynamics of cell fate decisions. To enable comparisons across conditions, the number of initial clones was normalized to be 100 in each condition. At least 18 independent clones were tracked for each condition from at least two independent experimental replicates.

Article Snippet: PGE2 (10 ng/mL, Cayman Chemical, Ann Arbor, MI) or OSM (100 ng/mL, R&D Systems, Minneapolis, MN) was then administered, and time lapse imaging was started.

Techniques: Clone Assay