Unifilter plates

Kinase selectivity profiling was performed through contract research services at the MRC PPU International Centre for Kinase Profiling, Dundee, UK., using their radioactive filter binding assay methodology as previously described (Hastie et al., 2006 (link)). Briefly, BMS-754807 (AstraZeneca) was tested at a single concentration of 1 μM, in duplicate, against 76 kinases. BMS-754807 was pre-incubated at room temperature for 5 min in the presence of 5-20 mU of kinase enzyme and the respective peptide/protein substrate, before initiation of the reaction by addition of ³³P-γ-ATP. Assays were incubated at room temperature for 30 mins before termination by the addition of 50 mM orthophosphoric acid. Assay plates were harvested onto P81 Unifilter plates (Whatmann) by a Packard Harvester (PerkinElmer) using a wash buffer of 50 mM orthophosphoric acid and subsequently dried in air. The dry Unifilter plates were then sealed on the addition of MicroScint (PerkinElmer) and counted using a Topcount NXT scintillation counter (PerkinElmer). Data was reported as a % incorporation of radiolabelled phosphate from ³³P-γ-ATP into the peptide or protein substrate in relation to the maximal enzyme control.

Competition binding was performed in duplicate in the wells of a 96‐well plate containing binding buffer (optimized for each receptor), cell membrane extracts (approximately 20,000 cells distributed in the 96‐well plate), radiotracer, and test agonist. Non‐specific binding was determined by co‐incubation with 200‐fold excess of competitor. Cells were incubated and exposed to varying concentrations (1 pM to 10 μM) of a range of displacer agonists (see below). The samples were incubated in a final volume of 0.1 ml and then filtered over Unifilter plates (Perkin Elmer, Massachusetts, USA) pretreated for 2 hr to limit tracer non‐specific binding. Filters were washed five times with 0.5 ml of ice‐cold washing buffer (tris 50 mM pH 7.4) and 50 μl of Microscint 20 (Perkin Elmer) were added to each filter. The plates were incubated 15 min at room temperature on an orbital shaker and then counted with a TopCount™ (Perkin Elmer) for 1 min per well.

PBMC at ≤10⁶ cells/mL in growth medium were incubated in quadruplicate wells of 96-well microplates (Corning) at 100μL/well in the presence of 100 μL of inactivated CMV-infected cell lysate, inactivated varicella zoster virus (VZV)-infected cell lysate, C. albicans antigen (Greer), HIV inactivated virion [26 (link)], the appropriate mock-infected cell control antigens and phytohemagglutinin (PHA; Sigma) positive control. After 6 days of antigenic or mitogenic stimulation at 37°C, in a 5% CO₂ and 95% H₂O atmosphere, cells were pulsed with 1 μCi of ³H-thymidine and harvested 6 h later onto Unifilter plates (Perkin Elmer). Radioactivity gathered on the filters was counted in scintillation fluid (Perkin Elmer) with a microplate scintillation counter (Packard). Results were expressed in median counts per minutes (cpm) of the quadruplicate wells. Inhibition was measured on samples that showed ≥3-fold increase of median cpm in antigen- compared with mock-stimulated wells.
Treg expansion with rhIL2 (30ng/mL) was carried out in 96-well microtiter plates as described above for up to 15 days, during which rhIL2 and growth medium were replenished twice weekly. At the end of the expansion, viable cells were counted with a Guava 8HT instrument.

YFP⁺ Treg cells were isolated by sorting lymph node samples pre-enriched for CD25⁺ cells using a mouse CD25 MicroBead Kit (Miltenyi Biotec) on a FACSAria (BD Biosciences). CD4⁺CD25⁻ Tconv cells were isolated from the CD25⁻ fraction obtained from the same samples by negative selection using FITC-conjugated Abs and anti-FITC Microbeads (Miltenyi Biotec) as described above (T cell isolation). Treg cells were cocultured in known ratios with 1 × 10⁵ Tconv cells per well in 96-well round-bottom Nunclon plates (Nunc) and stimulated with 2 × 10⁴ anti-CD3/anti-CD28–coated Dyna Beads (Dynal). After 96-h incubation at 37°C in an atmosphere of 5% CO₂, proliferation was measured by [³H]thymidine incorporation. [³H]thymidine was added at 0.5 μl per well, and plates were incubated for 6 h before cells were harvested to UNIFILTER Plates (PerkinElmer) using a Tomtec 96 Harvester. Collection plates were air dried overnight and 30 μl MicroScint-20 (PerkinElmer) added to each well. A TopCount Scintillation Counter (PerkinElmer) was then used to read the relative incorporation of [³H]thymidine in each well.

Cells were cultured in 96-well flat-bottom plates (Nalge Nunc International, Naperville, IL, USA) at 1 × 10⁵ cells/well and 10% CD14⁺ myeloid cells (as indicated) in a final volume of 200 μL culture medium with triplicate wells. Cells were incubated for 72 h followed by the addition of 0.4 μCi ³H-thymidine (Amersham Pharmacia Biotech)/well for another 16 h of culture. Cells were freeze–thawed and harvested onto Unifilter plates (Perkin–Elmer, Boston, MA, USA) and incorporation of ³H-thymidine was measured as counts per minute (cpm) using a liquid scintillation counter (Top-Count, Perkin–Elmer). Each experiment was performed separately with cells isolated from several animals as indicated.