Recombinant mouse egf

Esophageal keratinocytes were isolated from vehicle-treated or 4NQO-treated K5Cre^ERT2;R26tdTomato^lsl/lsl or Notch1^loxP/loxP (Jacskon Laboratories) mice under an IACUC-approved protocol as described previously^{65 (link), 84 (link), 85 (link)}. Using 24-well plates, 5000 cells were seeded per well in 50 μl Matrigel. After solidification, 500 μl of DMEM/F12 supplemented with 1× Glutamax, 1× HEPES, 1× N2 Supplement, 1× B27 Supplement, 0.1 mM N-acetyl-L-cysteine (Sigma-Aldrich), 50 ng/ml mouse recombinant EGF (R&D Systems), 2.0% Noggin/R-Spondin-conditioned media and 10 μM Y27632 (Tocris Biosciences, Bristol, UK) were added and replenished every other day. For ex vivo recombination, organoids were cultured in the presence of Adenovirus vector containing Cre recombinase and GFP (University of Iowa Gene Transfer Vector Core). Adenovirus vector containing GFP alone was used as a control. Adenovirus vectors were used at 1:500 at the time of organoid plating. Organoid formation rate was calculated as the percentage of the number of organoids formed at day 7 per total number of cells seeded at day 0. After 14 days organoids were recovered by digesting Matrigel with Dispase I (BD Biosciences, San Jose, CA; 1 U/ml) and fixed overnight in 4.0% paraformaldehyde. Specimens were embedded in 2.0% Bacto-Agar: 2.5% gelatin prior to paraffin embedding.

Both WT and Npc1^−/− P7 microglia were cultured at a density of 5 × 10⁵ cells/well in a 12-well plate in microglia culturing medium. At 5DIV, microglia culturing medium was replaced with serum-free medium, containing only DMEM/F12 and 1% PenStrep. After 3 h of serum deprivation (t = 0), cells were treated with mouse recombinant EGF (40 ng/ml, R&D System) and cycloheximide (20 µg/ml, Sigma Aldrich) in serum-free medium. Cells were lysed at different time points (0, 1, 3, and 6 h) after treatment in STET lysis buffer and supplemented with protease and phosphatase inhibitors. EGF receptor (EGFR) level in each lysate was analyzed via western blot using an antibody against EGFR (anti-EGFR antibody EP38Y, Abcam). Each experimental group was tested in microglia isolated from three independent experiments (n = 3).

Esophageal keratinocytes were isolated from untreated, control treated or 4NQO- treated mice. Using 24-well plates, 5000 cells were seeded per well in 50 μl Matrigel. After solidification, 500 μl of DMEM/F12 supplemented with 1× Glutamax, 1× HEPES, 1× N2 Supplement, 1× B27 Supplement, 0.1 mM N-acetyl-cysteine (Sigma-Aldrich), 50 ng/ml mouse recombinant EGF (R&D Systems), 2.0% Noggin/R-Spondin-conditioned media and 10 μM Y-27632 (Tocris Biosciences, Bristol, UK) were added and replenished every other day. Organoid formation rate was calculated as the percentage of the number of organoids formed at day 7 per total number of cells seeded at day 0. After 10 days, the organoids were recovered by digesting Matrigel with Dispase I (BD Biosciences; 1 U/ml) and fixed overnight in 4.0% paraformaldehyde. Specimens were embedded in 2.0% Bacto Agar and 2.5% gelatin prior to paraffin embedding. Cross sections (50 µm) of the organoids were stained with hematoxylin-eosin. Immunohistochemistry using antibodies (as indicated) was performed on parallel sections.