alp alkaline phosphatase sk 5100 vector laboratories  (Vector Laboratories)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer

doi: 10.1016/j.jcmgh.2017.10.007

Figure Lengend Snippet: Modulation of in vitro human crypts by SCFAs. ( A ) Biochemical gradients applied to the tissue. ( B ) Side view of representative crypts from the arrays ( scale bar : 100 μm) under different SCFA gradients: no SCFA gradient (control), acetate (0–15 mmol/L), propionate (0–5 mmol/L), and butyrate (0–5 mmol/L). Red, ALP; green, EdU-based stain; blue, DNA. ( C ) Top view of human crypt array. Upper panel scale bar : 1 mm; lower panel scale bar : 100 μm. ( D ) Number of EdU + cells per crypt under different gradients. ( E ) Relative proliferation length, defined as the length of EdU + crypt over the total length of the crypt for the different gradients. ( F ) Normalized ALP activity for the various SCFA gradients. ( G and H ) EdU incorporation and ALP activity under different butyrate gradients. Basal butyrate (0 mmol/L) with the luminal butyrate concentration listed on the x-axis. N, noggin; R, R-spondin; W, Wnt-3A. ∗ P < .05 and ∗∗ P < .005.

Article Snippet: Cells were first pulsed with 10 μmol/L of EdU for 24 hours at 37°C, rinsed with PBS, and incubated with a red ALP substrate (SK-5100; Vector Laboratories, Burlingame, CA) in Tris buffer (0.15 mol/L, pH 8.4) for 30 minutes at 37°C.

Techniques: In Vitro, Staining, Activity Assay, Concentration Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer

doi: 10.1016/j.jcmgh.2017.10.007

Figure Lengend Snippet: The effect of the cytokines, IFN-γ and TNF-α, on in vitro human crypts. ( A ) Biochemical gradients applied to the tissue. ( B ) Side view of representative crypts from the arrays under gradients of vehicle (0.1% BSA, control), or cytokines of 10 ng/mL IFN-γ and 100 ng/mL TNF-α. Scale bar : 100 μm. Red, ALP; green, EdU-based stain; blue, DNA. ( C ) Confocal 3-dimensional reconstruction of human crypt array viewed from basal side. Upper panel scale bar : 1 mm; lower panel scale bar : 100 μm. ( D ) Effect of cytokine gradient on number of EdU + cells per crypt (N = 20 crypts). ( E ) Box plot showing the relative position of EdU + cells along the basal–luminal axis of crypts. Zero represents the basal end of the crypt and 1 marks the luminal end of the crypt. ( F ) Normalized ALP stain for the array with the focal plane at the luminal side with a depth of field of 60 μm (N = 4 arrays). ( G ) A cross-sectional image through an in vitro crypt obtained by confocal fluorescence imaging. Left : Crypt in the absence of a cytokine gradient. Right : Crypt in the presence of an IFN-γ/TNF-α gradient. The cells were stained with Hoechst 33342 (blue) and propidium iodide (PI) (red). Scale bar : 100 μm. ( H ) Box plot depicting the number of dead cells per cross-section of crypt (N ≥ 24). Unpaired 2-tailed Student t test: * P < .05; ** P < .005. N, noggin; R, R-spondin; W, Wnt-3A.

Article Snippet: Cells were first pulsed with 10 μmol/L of EdU for 24 hours at 37°C, rinsed with PBS, and incubated with a red ALP substrate (SK-5100; Vector Laboratories, Burlingame, CA) in Tris buffer (0.15 mol/L, pH 8.4) for 30 minutes at 37°C.

Techniques: In Vitro, Staining, Fluorescence, Imaging