Microbeta liquid scintillation counter

Cells were seeded in 96-well plates (Corning, Corning, NY) for 24 h prior to treatment. HCT116 cells were treated at ~70% confluency and then cultured for an additional 48 h. IC1 cells (5000 cells/well), MET1 cells (2500 cells/well), and primary keratinocytes (3750 cells/well) were treated as indicated and cultured for an additional 72 h. Six hours prior to harvesting cells, 10 µl of a 0.1 mCi ml⁻¹ [³H]thymidine (American Radiolabeled Chemicals, St. Louis, MO) solution in media was added to each well. Cells were harvested using a Filtermate Harvester (PerkinElmer, Waltham, MA) and radioactivity was measured using a MicroBeta liquid scintillation counter (PerkinElmer, Waltham, MA). Data represent three independent experiments with four technical replicates per experiment. Where p-values are reported, the unpaired t test was used to determine significance.

Mice were vaccinated s.c. with 10 µg of inactivated CHIKV as described [48] (link). Standard proliferation assays using tritiated thymidine uptake were undertaken using splenocytes isolated 3 weeks post vaccination. Briefly, splenocytes (2.5×10⁵ cells/96 well, 6 replicates) were cultured with 10 µg/ml of inactivated CHIKV [48] (link) for 3 days, tritiated thymidine was then added and cells harvested the next day onto a MicroBeta Filtermat-96 A using the FilterMate™ Cell Harvester (PerkinElmer). Radioactivity was measured using the MicroBeta Liquid Scintillation Counter (PerkinElmer).

Increasing concentrations of nanobodies or unlabeled CX3CL1 or CCL5 were added to 100 pM ¹²⁵I-CX3CL1 or 200 pM ¹²⁵I-CCL5 in HEPES binding buffer (50 mM HEPES-HCl, pH 7.4; 1 mM CaCl2; 5 mM MgCl2; 0.1 M NaCl; 0.5% (w/v) bovine serum albumin). Radioligand alone and radioligand + 100 nM of unlabeled CX3CL1 or CCL5 were used as controls for total binding and non-specific binding. A total of 3 µg of HEK293T, HEK293T-iUS28 or HEK293T cells transfected with the US28 receptor were added to the ligands and incubated for 2 h at room temperature. Membranes were harvested on 0.5%(w/v) PEI-soaked GF/C filter plates (Perkin-Elmer, Waltham, MA, USA) and dried for 30 min at 60 °C. In addition, 100 pM of ¹²⁵I-CX3CL1 or 200 pM ¹²⁵I-CCL5 was spotted on the GF/C filter plate to determine radioligand concentration. Scintillation fluid MicroScint-O (Perkin-Elmer) was added to the GF/C filter plate and radioactive decay was measured using a Microbeta liquid scintillation counter (Perkin-Elmer). Data were analyzed using GraphPad Prism version 8.0.