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