mouse esc line d3  (Thermo Fisher)

Journal: PLoS ONE

Article Title: Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency

doi: 10.1371/journal.pone.0043708

Figure Lengend Snippet: A, B. MPLSM single optical sections of mouse ESCs at different stages of differentiation (ESC and days 1, 3, 5, and 8 of EB formation; ESC: n = 24, 27, 14, 23, 13, 4 colonies, respectively; EB: n = 7, 16, 6, 18, 8, 4 EBs, respectively) using 780 nm excitation. A. Endogenous fluorescence intensity images following application of a background subtraction FIJI plugin macro (see ) to remove 90% of the background of the region of interest prior to intensity analysis. Complete timeline of images, including raw image data, is in . B. Color mapped lifetime values of MPLSM single optical sections of mouse ESCs at different stages of differentiation (mESC and days 1, 3, 5, and 8 of EB formation; ESC: n = 13, 20, 8, 18, 10, 18 colonies, respectively; EB: n = 7, 7, 6, 12, 7, 3 EBs respectively) using 780 nm excitation. Color bar indicates lifetime values for τ m . Detailed statistics in text and . Scale bars = 50µm. C. Background subtracted fluorescence levels were normalized by the mean background subtracted intensity per pixel of the mESCs for that day. For this, and all subsequent figures, bars are mean ± standard deviation. Horizontal lines indicate statistical difference ( P <0.05) between normalized EB intensity values (Dunn’s method pairwise multiple comparison following ANOVA). D. Quantitation of lifetime values for τ m of NADH (780 nm excitation). Values are normalized to corresponding mean mESC lifetime values. Horizontal lines indicate statistical difference ( P <0.05) between normalized mEB lifetime values (Dunn’s method pairwise multiple comparison following ANOVA).

Article Snippet: Mouse embryonic stem cell culture and embryoid body formation: Mouse ESC line D3 and a pOct4:GFP-expressing mESC cell line were maintained as pluripotent in DMEM (Invitrogen, Carlsbad, CA) or DMEM-Glutamax (Invitrogen) with 10% fetal bovine serum (Invitrogen), 1X MEM non-essential amino acids (Invitrogen), 6 µl/100 ml β-mercapto-ethanol (Sigma, St. Louis, MO), and penicillin/streptomycin (50 U penicillin and 50 µg/ml streptomycin) (Invitrogen).

Techniques: Fluorescence, Standard Deviation, Quantitation Assay

Journal: PLoS ONE

Article Title: Foxm1 Mediates LIF/Stat3-Dependent Self-Renewal in Mouse Embryonic Stem Cells and Is Essential for the Generation of Induced Pluripotent Stem Cells

doi: 10.1371/journal.pone.0092304

Figure Lengend Snippet: (A) The upregulation of FOXM1 expression maintained the typical mESC morphology and positive alkaline phosphatase staining in D3 ES cells in the absence of LIF and feeder layers. A lentiviral vector expressing human FOXM1B was produced to infect D3 ES cells and the lentivirus-infected D3 ES cells were cultured for one week in the absence of LIF and feeder layers. Pictures were taken at 200× magnification using a TE2000 microscope (Nikon). Alkaline phosphatase staining (AP) was performed in D3 ES cells, D3 ES cells or FOXM1-overexpressing D3 ES cells cultured without LIF and feeders for one week. The percentage of differentiated, mix, or undifferentiated colonies of different D3 ES cell samples was calculated. (B) The FOXM1 upregulation recovered the levels of Oct4 and Nanog in D3 ES cells in the absence of LIF and feeder layers. Western blot analyses were performed for the expression of Foxm1 (FOXM1), Stat3, phosphorylated Stat3 (p-Stat3), Oct4, Nanog, GFP, and β-actin in D3 ES cells, D3 ES cells without LIF and feeders for one week, or FOXM1-overexpressing D3 ES cells without LIF and feeders for one week. (C) The effects of FOXM1 overexpression on the mRNA levels of pluripotency-related genes in D3 ES cells without LIF and feeders at passage 5. Quantitative RT-PCR analyses were performed for Foxm1, Utf1, Oct4, Nanog, Esrrb, Klf4, Tbx3, Klf2 and Sall4 mRNA levels. The levels of each transcript in D3 ES cells were set at 1.0. Error bars indicated standard deviation (n = 3). (D) The typical mESC morphology and positive alkaline phosphatase staining were maintained in FOXM1 overexpressing D3 ES cells during long-term culture without LIF and feeders. Pictures were taken for FOXM1 overexpressing D3 cell culture without LIF and feeders at passage 5, passage 7, and passage 9. The colonies of typical positive alkaline phosphatase staining were shown in the squares of each picture. (E) The teratomas formed by FOXM1 overexpressing D3 ES cells without LIF and feeders at passage 5 contained derivatives of all three germ layers. Sections of the formed teratomas were stained with hematoxylin and eosin dyes and the representative photographs were taken using a TE2000 microscope.

Article Snippet: Mouse ESC lines D3 (ATCC, USA) and iPSCs were co-cultured with feeder cells [MEFs (isolated from BALB/c mouse embryos at day 13 of gestation and expanded for 4 passages, 3×10 4 cells/cm 2 ) that were treated with 10 mg/L Mitomycin C (Sigma, USA) for 2.5 h before use] in 0.1% gelatin (Sigma, USA)-coated dishes at 37°C in 5% CO 2 .

Techniques: Expressing, Staining, Plasmid Preparation, Produced, Infection, Cell Culture, Microscopy, Western Blot, Over Expression, Quantitative RT-PCR, Standard Deviation