Fitc brdu flow cytometry kit

The conventional suppression assay was used herein [10 (link)] with the following modifications: Conventional Tregs (CD4+ CD25+) as well as CD4+ CD25⁻ T cells were enriched from freshly-isolated splenocytes over commercial isolation columns (MACS column specific for CD4+ CD25+ T-cells; Miltenyi Biotec, Bergisch Gladbach, Germany). For the suppression assay, Tregs were added at increasing dilution ratios (1:1–1:16) to CD3/CD28-stimulated syngeneic freshly isolated splenic- 1 × 10⁵ CD4+ CD25− T cells. CD3/CD28 stimulation was provided by the addition of Epoxy DynaBeads coated with anti-CD3 anti-CD28 monoclonal antibodies (Invitrogen, Carlsbad, CA). The incubation was carried out for 5 days in RPMI-1640 with 10% FBS. At the end of incubation, 1 mm of BrdU was added for the final 16 h to assess proliferation. Suppression was determined by the level of FITC fluorescence and percentage of cells fluorescent by FACS analysis using the FITC-BrdU flow cytometry kit (BD Biosciences, San Jose, CA). The experiment was performed in triplicate.

Human primary esophageal fibroblasts were obtained from esophageal mucosa of healthy donors (Arkansas Regional Organ Recovery Agency). For experiments analyzing cell proliferation, cells were untreated (phosphate buffered saline, PBS) or treated with 50 ng/ml of LIGHT and incubated with 10 μM BrdU for 24 hours prior to collection and staining (see flow cytometry section). The cells were permeabilized, fixed and stained using a FITC-BrdU flow cytometry kit according to manufacturer’s recommendations (BD Biosicences, Franklin Lakes, NJ). For analysis of IL-13 and LIGHT-mediated gene expression, cells were untreated (PBS) or stimulated with 50 ng/ml of either cytokine or a combination of both for 24 hours. After treatment, the cells were lysed in trizol and RNA extracted using a phenol-chloroform-based method. RNA sequencing was performed according to our previously described methods (18 (link), 19 (link)). Data from LIGHT-stimulated cells was previously submitted (GSE143482). Data from IL-13-stimulated fibroblasts can be found in GSE212731 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE212731). RNA was sequenced at the Genomics CORE in La Jolla Institute for Immunology.