Cfse cell division tracker kit

Spleen from OT-I mice were crushed through 70 um cell strainers with complete RPMI 1640. Splenocytes were pelleted and then treated with 0.8% NH₄Cl for 5 minutes to lyse red blood cells. CD8+ T cells were enriched using negative selection kit (Miltenyi, 130-096-495) per manufacture’s instruction. CD8+ T cells were counted and labeled with CFSE Cell Division Tracker Kit (Biolegend, 423801). Fully differentiated BMDMs were scraped off from culture dish and reseeded into 96 well plate at a density of 1 x 10⁵ cell per well. Two hours after seeding, BMDMs were pulsed with OVA_257-264 peptide at indicated concentrations for 2 hours, washed three times with RPMI 1640, and incubated with 1 x 10⁵ cell labeled OT-I CD8+ T cells for 5 days. Cell proliferation rate was determined by flow cytometry.
For intrahepatic T cell proliferation, NPCs were harvested from liver tissue and labeled with CFSE Cell Division Tracker Kit (Biolegend, 423801). Labeled cells were transferred to 96-well plates and stimulated with CD3/CD28 Dynabeads for up to 5 days. Cell proliferation rate was determined by flow cytometry.

Total splenocytes were labeled with the CFSE cell division tracker kit (BioLegend) and cells were cultured in RPMI containing 20% regular fetal calf serum with or without RA (20 nM), OCH (200 ng/ml) and IL-2 (100 u/ml) for 24h. The cultured cells were centrifuged through an underlay of newborn calf serum (100%) in for 7 min and were further cultured without OCH but with IL-2 for 3 additional days in the presence or absence of RA. Cells were harvest and analyzed for the CFSE intensity of CD1d-Tet⁺ cells by flow cytometry.