5 bromo 2 deoxy uridine brdu cell proliferation assay

We used the DC-naïve T cell proliferation assay to evaluate the ability of Pmp18.1 to elicit helper CD4+ T cell proliferation. Splenocytes obtained from spleens of naïve mice using the gentleMACS Dissociator (Miltenyi Biotech, Auburn, CA) were resuspended in PBS containing BSA and EDTA. Purification of CD4+ T cells was achieved by positive selection using the MidiMACS system and labeled CD4 (L3T4) mouse microbeads (Miltenyi Biotech, Auburn, CA). Purity of the CD4+ T cells was analyzed by flow cytometry and shown to be at least >95%. Purified naïve CD4⁺ T cells (2 × 10⁵ cells/well) were cultured in 96-well plates (Corning Glass Work, Acton, MA) with BMDCs (2 × 10⁵ cells/well) and either Pmp18.1 (10 μg/ml) or Pmp18.1 plus VCG (100 μg) in 200 μl of C-RPMI medium supplemented with GM-CSF at 37°C in 5% CO₂. Control cultures containing DCs and naïve T cells without antigen served as internal control. After 7 days of culture, T cell proliferation was assessed essentially as described previously (Eko et al., 2011a (link)) using the 5-Bromo-2′-deoxy-uridine (BrdU) cell proliferation assay (Roche Molecular Biochemicals, Indianapolis, IN). Absorbance values were read at 450 nm and the ratio between stimulated and non-stimulated cells (stimulation index, SI) was then calculated.

The ability of purified immune T cells to proliferate in response to in vitro restimulation in culture with Pmp18.1 was assessed using the 5-Bromo-2’-deoxy-uridine (BrdU) cell proliferation assay according to the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN) and described previously (30 (link)). Briefly, gamma-irradiated (2000 rad) splenocytes (10⁶/ml) purified from naive animals were co-cultured with purified T cells (10⁶ cells/ml) and 5 μg/ml of rPmp18.1 at 37°C in 5% CO₂. After 3 days, the plates were incubated with BrdU labeling solution for 18 h followed by incubation with peroxidase labeled anti-BrdU antibody for 1 h at 37°C. Plates were then developed with 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate for 30 min and BrdU incorporation was detected using a scanning multi-well spectrophotometer (Spectra-Max 250 ELISA reader, Molecular Devices, Sunnyvale, CA). The stimulation index (SI) was calculated as the ratio between stimulated and non-stimulated cells for triplicate cultures. The experiment was repeated twice for confirmation.