n2b27 base  (MedChemExpress)

Journal: Biology Open

Article Title: An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

doi: 10.1242/bio.20148409

Figure Lengend Snippet: (A) Sox1::GFP (left) or T::GFP (right) mESCs exposed to serum and LIF (SL), 2i and LIF (2i + L), N2B27 or Activin and Chi (AC) for the indicated durations and GFP expression analysed by flow cytometry. Hashed vertical line bisecting the population profile plots indicates the peak maximum of the negative control. (B) T::GFP mESCs differentiated in AC for 2 days (0–2), immunostained for Hoechst, Oct4 and Sox2 and imaged by confocal microscopy. (C) TNGA mESCs cultured in SL and FACS sorted into low- (indicated in pink) and high- (indicated in dark blue) expressing populations (top) were replated in AC conditions for 2 days, immunostained for Brachyury and analysed by confocal microscopy (bottom). TNGA cells cultured in SL conditions served as a negative control for Brachyury immunostaining. (D) T::GFP (red) and Sox1::GFP (blue) mESCs were differentiated for 2 days in AC conditions after exposure to N2B27 for the indicated durations. GFP expression was measured by flow cytometry. (E) T::GFP (Red) or Sox1::GFP (black) mESCs plated and treated with N2B27 for 6 days with single, 1-day pulses of AC on days indicated above graphs. For the control, T::GFP or Sox1::GFP cells were incubated with 6 days of either AC or N2B27, respectively. Grey bar indicates period of AC pulsing. (F) Sox1::GFP mESCs treated with 1 ng/ml BMP4 for durations indicated or 5 days (0–5) in N2B27. GFP expression measured by flow cytometry and fluorescence values displayed are normalised to the N2B27 control. (G,H) Live-cell imaging of TNGA cells in N2B27 alone (G) or supplemented with 1 ng/ml BMP4 (H). Scale bars: 100 µm.

Article Snippet: To obtain serum-free pluripotency conditions, cells were cultured in 2i + LIF; 2i uses an N2B27 base medium (NDiff 227, StemCells Inc., UK) supplemented with 1 μM PD0325901, 3 μM Chi and LIF.

Techniques: Expressing, Flow Cytometry, Negative Control, Confocal Microscopy, Cell Culture, Immunostaining, Control, Incubation, Fluorescence, Live Cell Imaging

Journal: Biology Open

Article Title: An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

doi: 10.1242/bio.20148409

Figure Lengend Snippet: (A,B) Proportion of GFP positive cells in Sox1::GFP (A) or T::GFP (B) cells exposed to the indicated factors for 5 days. (C,D) Proportion of GFP positive cells in Sox1::GFP (C) or T::GFP (D) cells exposed to the indicated factors for 2 days prior to switching medium to N2B27 for 3 days (A, Sox1::GFP) or AC conditions for 2 days (B, T::GFP). Expression of GFP was measured by flow cytometry. All data presented here are normalised to 5 days in N2B27 (Sox1::GFP) or 2 days N2B27 followed by 2 days AC conditions (T::GFP).

Article Snippet: To obtain serum-free pluripotency conditions, cells were cultured in 2i + LIF; 2i uses an N2B27 base medium (NDiff 227, StemCells Inc., UK) supplemented with 1 μM PD0325901, 3 μM Chi and LIF.

Techniques: Expressing, Flow Cytometry

Journal: Biology Open

Article Title: An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

doi: 10.1242/bio.20148409

Figure Lengend Snippet: (A,B) Population profiles of cells analysed by flow cytometry from either (A) Nanog::GFP (TNGA) or (B) Rex1::GFP mESCs after 2 (0–2) or 3 (0–3) days in the medium indicated (SL, 2iL, N2B27, Chi, XAV939, Activin (Act), SB43, BMP, DM-H1). Population in grey is non-fluorescent control and hashed vertical lines correspond to the peak maximum of the fluorescence from cells in either N2B27 (a,c) or SL (b,d) conditions at each time-point. (C) Live-cell imaging of TNGA mESCs differentiated for 96 h in N2B27, Chi, Activin or BMP. (D) Live-cell imaging of TNGA mESCs differentiated for 96 h in either SB43 or XAV939. (E) Sox1::GFP cells were grown in the indicated medium for 2 days and a further day in N2B27 prior to RNA extraction and RT-qPCR analysis for the indicated genes. Data normalised to the house-keeping gene Ppia ( supplementary material Fig. S2 ) and displayed as values higher, similar or lower than the SL control. Scale bars (in all live-cell imaging experiments): 100 µm.

Article Snippet: To obtain serum-free pluripotency conditions, cells were cultured in 2i + LIF; 2i uses an N2B27 base medium (NDiff 227, StemCells Inc., UK) supplemented with 1 μM PD0325901, 3 μM Chi and LIF.

Techniques: Flow Cytometry, Control, Fluorescence, Live Cell Imaging, RNA Extraction, Quantitative RT-PCR

Journal: Biology Open

Article Title: An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

doi: 10.1242/bio.20148409

Figure Lengend Snippet: (A–C) T::GFP (A) and Sox1::GFP (B,C) cells were grown in N2B27 for 2 days prior to transferring to the indicated conditions for 2 days (T::GFP) or 3 days (Sox1-GFP). GFP expression was assessed by flow cytometry and data normalised to control conditions (0–2 days N2B27, 2–4 days AC for T::GFP, 0–5 days N2B27 for Sox1::GFP) and presented in bar charts. (D) Sox1::GFP cells were plated in either N2B27, SB43 or XAV939 for the first 2 or 3 days of culture and then switched to N2B27, Activin (Act) or BMP4 for the remaining period. GFP expression was assessed by flow cytometry on day 5. Data are displayed as a bar chart and have been normalised to control conditions (0–5 days N2B27).

Article Snippet: To obtain serum-free pluripotency conditions, cells were cultured in 2i + LIF; 2i uses an N2B27 base medium (NDiff 227, StemCells Inc., UK) supplemented with 1 μM PD0325901, 3 μM Chi and LIF.

Techniques: Transferring, Expressing, Flow Cytometry, Control

Journal: Biology Open

Article Title: An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

doi: 10.1242/bio.20148409

Figure Lengend Snippet: Mouse ES cells cultured in LIF and BMP were transferred to N2B27 and allowed to differentiate for 5 days. (A) Average (bulk) expression levels of several genes associated with pluripotency, NECT and PS fate restriction, as well as targets for Wnt/Notch, Nodal/BMP and FGF/RAR signalling, were measured daily using fluidigm quantitative RT-PCR. Colour-coded values indicate the log-fold change values with respect to the initial LIF + BMP condition expression levels measured by RT-PCR and normalised to a housekeeping gene. (B) Principal Component Analysis of the bulk expression profiles of 34 genes for 2i, LIF and BMP and the 5 days of differentiation in N2B27 normalised to Gapdh. Each single point indicates a technical repeat. (C) Loadings plot of the PCA indicating the genes that contribute the most to the first two components. (D,E) Expression levels of the 29 most relevant genes of 90 analysed using fluidigm technology and single-cell RT-PCR over the 5-day differentiation experiment (see main text and supplementary material Fig. S3 for details). (D) Heat maps of single-cell log2-expression levels normalised to Gapdh (each row represents an individual cell) for the most significant genes. Genes (in columns) were clustered according to the pairwise similarities in their expression levels. Additional genes of interest are also displayed in the right-most panels. Data for 2i and LIF conditions are also shown on the panel on top. (E) Violin plots of the data in panel D showing kernel density estimates of the mRNA distribution (pale red and blue areas) together with single cell expression levels (black dots), the population average (blue line) and median (red dot).

Article Snippet: To obtain serum-free pluripotency conditions, cells were cultured in 2i + LIF; 2i uses an N2B27 base medium (NDiff 227, StemCells Inc., UK) supplemented with 1 μM PD0325901, 3 μM Chi and LIF.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction