Wallac microbeta trilux

K562 target cells (10⁶) were labelled with 100 µCi of Na₂⁵¹CrO₄ (Perkin Elmer, Waltham, MA, USA) for 1 h at 37 °C, 5% CO₂. Labelled cells were washed three times in medium and resuspended in NK cell coculture medium medium. Target cells were co-incubated with sorted eGFP⁺ NK cells at E:T ratios of 3, 1, 0.3, 0.1, and 0.03. Spontaneous release was measured by incubating target cells with medium alone, while maximum release was measured by incubating target cells in 1% Triton X-100. After 4 h, the supernatant was harvested and mixed with scintillation fluid (Perkin Elmer). Radioactivity was measured with a 1450 LSC&Luminescence Counter (Wallac Microbeta Trilux, Perkin Elmer). The mean percentage of cytotoxic activity of triplicates was calculated.

Rat resident peritoneal cells were obtained by injection of sterile PBS into the peritoneal cavity. Collected cells were centrifuged, resuspended in Serum-Free Medium (SFM, Life Technologies, Inc) and counted. Macrophages were obtained by adhering cells for 1 h at 37°C and 5% CO₂. After 1 h, non-adherent cells were removed by gentle washing with SFM and SFM medium supplemented with 20% of L929-conditioned media added. After 24 h, parasites were added to macrophages at a ratio of 3:1 for 1 h. After washing to eliminate extracellular parasites, cells were cultured for 40 h in the presence of [3H] uracil (5 μCi per well, Ci = 37 GBq) as previously described (Pfefferkorn and Pfefferkorn, 1977 (link)). Monolayers were washed three times in PBS, disrupted with 500 μl of lysis/scintillation solution (Optiphase Supermix, Perkin Elmer) and radioactivity measured by liquid scintillation counting using a Wallac MicroBeta TriLux (Perkin Elmer).

The binding experiments were carried out in 96-well plate format in a final volume of 200 μL as described earlier [21 (link)] with some modifications. In all assays, 20 μL membrane suspension was added to wells containing 140 μL binding buffer and 20 μL of competitive ligand (different concentrations of PRE-084 or 10 μM haloperidol (nonspecific binding) or binding buffer (control)) and incubated for 10 min at room temperature. Then, 20 μL [³H](+)-pentazocine was added (to achieve a final concentration of 2 nM for competitive binding experiments and 0.28 to 36 nM for saturation binding curve), and samples were incubated for 180 min at 37°C with continuous shaking. The bound radioligands were collected by rapid vacuum filtration through Millipore GF/C filters (Merck Millipore, Billerica, USA) presoaked in 0.3% polyethyleneimine. The filters were washed three times with 0.25 ml of 10 mM Tris buffer (pH = 8.0, 4°C), and their radioactivity was measured with a Wallac MicroBeta TriLux liquid scintillation counter (PerkinElmer, Waltham, USA).

K562 target cells (10⁶) were labeled with 100 µCi of ${\mathrm{Na}}_2^{\mathrm{51}}$ CrO₄ (Perkin Elmer, Waltham, MA, USA) for 1 h at 37°C, 5% CO_2. Labeled cells were washed three times in medium and resuspended in NK cell coculture medium. Cells were co-incubated with sorted eGFP⁺ NK cells at E:T ratios of 3, 1, 0.3, 0.1 and 0.03. Spontaneous release was measured by incubating target cells with medium alone, while maximum release was measured by incubating target cells in 1% Triton X-100. After 4 h, supernatant was harvested and mixed with scintillation fluid (Perkin Elmer). Radioactivity was measured with a 1450 LSC&Luminescence Counter (Wallac Microbeta Trilux, Perkin Elmer). The mean percentage of cytotoxic activity of triplicates was calculated.

MLR was performed according to Ratta et al.^{18 (link)} and was partly described earlier^{19 (link)}. In short, U-DCS cells were left untreated, incubated with a multiplicity of infection (MOI) of 5 of human cytomegalovirus (HCMV) or stimulated with 100 µg/ml Tetanus toxoid (Statens Serum Institut, Copenhagen, Denmark). Cells were collected 1 day later, extensively washed, irradiated (50 Gy) and plated in decreasing numbers with previously cryo-preserved allogenic peripheral blood mononuclear cells (PBMC) isolated from three HCMV-seronegative and three HCMV-seropositive (tested by Vidas CMV IgG, Biomerieux, France) buffy-coats, according to standard Ficoll density gradient protocols. PBMC were thawed, washed, resuspended in RPMI containing 5% human AB serum (Institut für Klinische Transfusionsmedizin und Immungenetik Ulm GmbH) and plated in triplicate at 1 × 10⁵ cells per well in a 96-well-U-bottom plate (Corning, NY). Increasing ratios of stimulators U-DCS and responder PBMC (stimulators: responders from 1:513 to 1:8) were co-cultured for 5 days and subsequently pulsed with 1 μCi/well [³H] thymidine for 18 h. Proliferation was determined by measuring the [³H] thymidine incorporation in a β-counter (Wallac MicroBeta TriLux, Perkin Elmer, Rodgau, Germany) and calculating the stimulation index (SI) as follows: SI = counts per minute (cpm) (PBMC + stimulators) / cpm (PBMC alone).

K562 target cells were labelled with Na₂CrO₄ (Bruno et al., 2014 (link); Perkin Elmer, Waltham, MA). eGFP⁺ NK cells (CD45⁺CD94⁺CD56⁺) were sorted and incubated in the presence of Bruno et al., 2014 (link) Cr-labeled K562 cells at variable effector-target ratio’s (E:T) in triplicate for 4 hr. Thereafter, the supernatant was mixed with scintillation fluid and the signal was measured using a 1450 LSC&Luminescence Counter (Wallac Microbeta Trilux, Perkin Elmer). The mean percentage of specific lysis was determined.

NIH3T3 and NIH-RAS cells were seeded at the density of 3000 cells/cm² and incubated overnight at 37°C and 5% CO₂, then medium change was done after 18 h. At 48 h (exponential growth condition) and 72 h (confluent growth condition), standard medium was replaced with 0.4 ml labeling medium (cysteine and methionine-free medium + 0.025 mCi/ml ³⁵S-Met, PerkinElmer), that was removed after 20-40-60 minutes or 30-50-60 minutes at 37°C and 5% CO₂. Cells were then washed once with cold PBS and scraped after adding lysis buffer. Cell lysates were centrifuged and an aliquot spotted on Whatman Glass Microfiber filters (Sigma Aldrich Inc.). To the remaining volume, 1 volume of cold 20% TCA (Sigma Aldrich Inc.) was added and, after 30 minutes in ice, samples were spotted on filters and washed twice with cold 10% TCA and ethanol (Sigma Aldrich Inc.). Air-dried filters were transferred to vials containing Ultima Gold MV scintillation fluid (PerkinElmer) and radioactivity measured in a beta-counter (Wallac Microbeta Trilux, PerkinElmer). Averages of technical triplicates for cell lysates (representing amino acid uptake) were calculated and the resulting values were normalized on total protein content, measured by using QuantiPro^TM BCA Assay Kit (Sigma Aldrich Inc.).