Topcount microplate scintillation counter

As described previously (O’Brien et al., 2010 (link)), 4 × 10⁵ spleen cells in culture medium (0.2 ml per well) were plated in 96 well U-bottom Corning Costar cell culture plates (Sigma–Aldrich). MOG_35-55 peptide was added to final concentrations of 1, 10, or 100 μg/ml. As a positive control, 1 μg/ml anti-CD3 and anti-CD28 antibodies (Beckman Coulter) were added. The equivalent volume of medium was added as a negative control. Cells were cultured for 72 h at 37°C with 5% carbon dioxide, pulsed with 1 μCi [³H]thymidine (Perkin Elmer, Cambridge, UK) per well, and cultured for a further 16 h. The cells were harvested onto glass fiber filter mats using a Packard harvester, and the plates were left to dry overnight. 25 μl of Microscint scintillation fluid (Perkin Elmer) was then added to each well before being assessed for thymidine incorporation using a liquid scintillation β counter (Top Count, Microplate Scintillation Counter; Packard, UK). Cell proliferation was recorded in counts per minute (CPM).

The cytolytic activity of T cell clones was assessed in 4 h ⁵¹Cr-release assays. Autologous or allogeneic melanoma cells or LCL received fresh medium two days prior to use as targets. Target cells suspended in 50 µl assay medium were loaded with 50 µl Na₂⁵¹CrO₄ (PerkinElmer Inc., Waltham, MA, USA) for 1 h. ⁵¹Cr-labeled targets were then washed three times with RPMI 1640 and combined with effector cells at ratios ranging between 1∶10 and 1∶3 (10³ targets per well) in duplicate or triplicate in a total volume of 150 µl assay medium containing a 50× target excess of unlabelled K562 cells. Maximum ⁵¹Cr release was defined by incubation of targets with assay medium containing 0.1% sodium dodecyl sulfate (Sigma-Aldrich), and spontaneous ⁵¹Cr release was determined by incubation of targets with assay medium alone. Plates were centrifuged at 50 g for 2 min, and then incubated for 4 h at 37°C. After incubation, 25 µl of supernatant was removed from each well, transferred to LumaPlates (Packard Bioscience, Lenexa, KS, USA) and allowed to dry overnight, prior to measurement as counts per minute (cpm) ⁵¹Cr release using a TopCount Microplate Scintillation Counter (Packard). Percent specific lysis was calculated using the standard equation:
Clones were considered to be specific if they lysed autologous targets at>15% of maximum lysis, and lysed allogeneic targets <5%.