Beta plate scintillation counter

CD4⁺ T cells were isolated from spleens of mice by magnetic activated cells sorting (Miltenyi Biotec, Auburn, CA). The CD4⁺CD25⁻, CD4⁺CD25⁺ subsets were further purified from the CD4⁺ T cells using a CD25⁺ Treg MACS isolation kit (Miltenyi Biotec). For proliferation assay, 1.0 × 10⁵ CD4⁺ cells were incubated in the presence of 1.0 × 10⁵ CD4⁻ cells (irradiated, used as antigen presenting cells) per well and stimulated with FVIII at 10U/ml (1U = 100 ng FVIII protein) for 72 hours, followed by adding 1μCi [³H]thymidine (PerkinElmer; Boston, MA) for the final 18 hours. [³H]thymidine incorporation was measured as counts per minute (c.p.m.) in a Betaplate scintillation counter (Perkin-Elmer). For suppressive assay, CD4⁺ T cells from mice treated with FVIII protein only were used as responders (Tresp) and CD4⁺CD25⁺ T cells from tolerized or naive mice at different time points were added as suppressor cells. To the co-culture of 0.8 × 10⁵ CD4⁺ T cells and 1.5 × 10⁵ antigen presenting cells, we added CD4⁺CD25⁺ T cells at indicated ratios. Suppression was calculated as[18 (link)]:
$$\%\text{suppression}=[1-(\mathrm{c}.\mathrm{p}.\mathrm{m}.(\text{CD}{4}^{+}\text{CD}{25}^{-}\mathrm{T}\text{cells}+\text{CD}{4}^{+}\text{CD}{25}^{+}\mathrm{T}\text{cells})/\mathrm{c}.\mathrm{p}.\mathrm{m}.\text{CD}{4}^{+}\text{CD}{25}^{-}\mathrm{T}\text{cells}\text{alone})]\times 100\%$$