alkaline phosphatase substrate kit  (Vector Laboratories)

Journal: bioRxiv

Article Title: Derivation of primed sheep embryonic stem cells and conversion to an intermediate naïve-like state

doi: 10.1101/2025.11.22.689923

Figure Lengend Snippet: Alkaline phosphatase staining of colonies grown in different matrices. B. Withdrawal of WNT inhibitors from culture condition led to differentiation, evidenced by the loss of colony definition and the emergence of morphologically distinct differentiated cells.

Article Snippet: Staining for ALP activity was performed using Vector Alkaline Phosphatase Substrate kit as per manufacturer instructions (Vector labs).

Techniques: Staining

Journal: bioRxiv

Article Title: Derivation of primed sheep embryonic stem cells and conversion to an intermediate naïve-like state

doi: 10.1101/2025.11.22.689923

Figure Lengend Snippet: (A–D) Flattened sESC EOS-OCT4-GFP cultures transferred into HENSM medium initially underwent substantial cell death, but by passages 3–5, compact EGFP-positive dome-shaped colonies emerged. (E–H) Dome-shaped colonies were independently generated from both primed lines (sESa and sESb), retained alkaline phosphatase staining, and expressed OCT4, SOX2, and NANOG. (I-L) Upon exposure to serum-containing differentiation medium, colonies flattened, lost ALP activity, and produced cells expressing markers of all three germ layers.

Article Snippet: Staining for ALP activity was performed using Vector Alkaline Phosphatase Substrate kit as per manufacturer instructions (Vector labs).

Techniques: Generated, Staining, Activity Assay, Produced, Expressing

Journal: bioRxiv

Article Title: Derivation of primed sheep embryonic stem cells and conversion to an intermediate naïve-like state

doi: 10.1101/2025.11.22.689923

Figure Lengend Snippet: A. Alkaline phosphatase staining of colonies grown in different naïve conditions. B. GFP-fluorescence intensity in Oct4-GFP colonies grown under different conditions. C. Karyotype of naïve like cells.

Article Snippet: Staining for ALP activity was performed using Vector Alkaline Phosphatase Substrate kit as per manufacturer instructions (Vector labs).

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: Inhibition of cytosolic DNA sensing and transposon activity safeguards pluripotency

doi: 10.1101/2025.03.26.645264

Figure Lengend Snippet: a) PCA of all genes in the indicated ESC samples, showing that IRF3 GOF samples cluster separately from all other conditions along PC1. RNA-seq data are from 4 replicates from independent experiments. b) Volcano plot of differentially expressed genes between IRF3 GOF versus BFP-only transfected ESCs. Blue points are significantly differentially expressed between the conditions (FDR ≤ 0.01). A set of pluripotency genes are indicated in purple, and the majority are down-regulated in IRF3 GOF samples. Selected IFN-I genes are shown in red (triangle for IRF3 indicates −log 10 FDR >25). c) The top 15 biological processes (BP) enriched in GO analysis of significantly downregulated genes in IRF3 GOF over BFP-only conditions (FDR < 0.05). d) Colony formation assays in ESCs after transfection of the indicated constructs and sorting. ESCs were plated at low density and cultured in the presence of Dox for 5 days before fixing and staining for alkaline phosphatase. Data are mean +/- s.e.m for n=3 wells, representative of 3-4 experiments. P values, one-way ANOVA with Šídák’s multiple comparisons test. e) Examples and quantification of immunofluorescence experiments for ESRRB and NANOG, 36 h after transfection with BFP or IRF3 GOF and FACS. ESCs were plated onto Matrigel immediately after sorting and fixed 1h later. P values, Welch’s t-test. f) As in e, but staining for OCT4 and SOX2. g) Morphology and alkaline phosphatase staining of parental KH2 ESCs or stable IRF3 GOF ESCs (clone B5, E7) showing the latter begin to lose undifferentiated morphology by 72 h after Dox addition. Data are representative of 3 clones and 2 experiments. h) Quantification of ESC differentiation status in g), according to AP staining. Colonies were scored according to the criteria of undifferentiated: >80% AP-positive, mixed: 30-80% AP-positive, differentiated: <30% AP-positive. Data shown are from 2 experiments. P values, Chi-squared test with Bonferroni correction for multiple comparisons. i) Colony formation assay in KH2 parental ESCs or IRF3 GOF ESCs (clone E7) 5 days after plating at low density in the presence or absence of Dox. All data in figure are representative of at least 2 independent experiments unless otherwise indicated.

Article Snippet: On the last day, ESCs were fixed with 4% PFA and stained with an Alkaline phosphatase kit (Vector, USA) according to the manufacturer’s protocol.

Techniques: RNA Sequencing, Transfection, Construct, Cell Culture, Staining, Immunofluorescence, Clone Assay, Colony Assay