Microbeta trilux counter

[³H]Ketanserin saturation binding assays were performed as previously reported [19 (link)] with minor modifications. [³H]Ketanserin binding (0.05–10 nM; eight concentrations) was used to calculate density (Bmax) and affinity (Kd) of 5-HT2AR. Non-specific binding was determined in the presence of the 5-HT2AR antagonist M100907 (1 μM). Competition curves of [³H]ketanserin binding (2 nM) with increasing concentrations of the agonist (±)-DOI (10⁻¹²–10⁻³ M) were also performed in order to delineate both the G-protein coupled and uncoupled 5-HT2AR conformations. Briefly, after incubation (60 min, 37 °C), free radioligand was discarded by rapid filtration under vacuum (1450 FilterMate Harvester, Perkin Elmer, Waltham, MA, USA). Filters were then rinsed, dried and bagged in Sample Bag with BetaPlate Scint scintillation cocktail. Radioactivity was detected by liquid scintillation spectrometry using a MicroBeta TriLux counter (Perkin Elmer, Waltham, MA, USA).

[³H]-8-OH-DPAT was used as a selective 5-HT_1AR agonist. The affinity (K_D) and maximal number of binding sites (B_max) were measured by saturation binding experiments over a radioligand concentration range of 0.1–14 nM. The affinity shift was determined by measuring the K_D obtained in saturation binding assays performed in the absence and presence of six concentrations of Zn (0.01–5 mM). Competition studies were performed with 2.5 nM of [³H]-8-OH-DPAT in the presence of various concentrations of orthosteric agonist serotonin in the absence and presence of Zn at two concentrations (10 and 500 μM). Non-specific binding was estimated in the presence of 10 μM 5-HT. The incubation buffer consisted of 50 mM Tris–HCl (pH 7.7), 10 mM MgCl₂, 10 mM pargyline and 0.1 % ascorbic acid. Radioligand binding assays were performed by incubating 30 μg of protein of the membrane suspension in 96-well microtitre plates for 60 min at room temperature with shaking, in a total volume of 200 μl. The binding reactions were stopped by filtration through GF/C Unifilter plates using a harvester (PerkinElmer). The plate filters were dried, and 20 μl of Ultima Gold MV (PerkinElmer) was added. Radioactivity was measured using a MicroBeta TriLux counter (PerkinElmer).

For a standard suppressor assay, CD4⁺FoxP3⁻ splenocytes (10⁵) were cultured in 96-well plates and activated with 1 μg soluble anti-CD3 (clone 2C11) antibody in the presence of APCs (5 × 10⁴ T cell depleted splenocytes) and titrating numbers of sorted CD4⁺FoxP3⁺ Tregs. Cultures were incubated for 72 h and pulsed with [³H]-Thymidine (0.5 μCi/well; Perkin Elmer) for the last 10 h. Incorporated isotope was measured by liquid scintillation counting (Micro Beta TriLux counter; Perkin Elmer). For a functional assay, spleen and LN suspensions were activated in vitro with 1 μg/ml soluble anti-CD3. After 72 h, proliferating cells were counted by Trypan blue exclusion using the Vi-cell XR cell counter (Beckman Coulter, Brea, CA).

Complete saturation binding assays were performed with [¹⁸F]altanserin (0.03–4 nM, eight concentrations), [³H]LSD (0.03–10 nM, ten concentrations) and [³H]MDL100907 (0.007–4 nM, ten concentrations) in order to determine the density (B_max) and the affinity (K_d) of 5­HT_2ARs. Incubation was carried out in tubes ([¹⁸F]altanserin) or 96-well plates ([³H]LSD and [³H]MDL100907) and started with the addition of the brain membrane preparation. Reactions were incubated for 40 min at 37 °C for [¹⁸F]altanserin binding assays and 90 min at 37 °C for [³H]LSD and [³H]MDL100907 binding assays. The presence of MDL100907 (1 µM) or altanserin (10 µM) was used to determine the non-specific binding of [¹⁸F]altanserin and [³H]LSD, and of [³H]MDL100907, respectively. After incubation, free radioligand was separated from bound radioligand by rapid filtration under vacuum through GF/C glass fibre filters pre-soaked with 0.5% polyethylenimine and counted for radioactivity gamma counting ([¹⁸F]altanserin; 2470 WIZARD2 Automatic Gamma Counter, PerkinElmer) or by liquid scintillation ([³H]LSD and [³H]MDL100907; MicroBeta TriLux Counter, PerkinElmer). Results were corrected for each radiotracer decay. Pairs of cases and controls were always processed at the same time and all samples were run in duplicate.

Alloantigen priming was performed using 10⁷ thymocytes isolated in PBS from BALB/c (H2^d) mice injected SQ into B6-FoxP3^RFP (H2^b) mice at the left lateral thoracic location. Post Vaccination Cyclophosphamide (PVCy) was given at 50 mg/kg/dose on days 3, 4, and 6 after BALB/c thymocyte SQ injections to the indicated treatment groups. Alloantigen exposed and PVCy treatment B6-FoxP3^RFPmice were euthanized at day 21 after alloantigen vaccination. Splenocytes were harvested to establish in vitro mixed lymphocyte reaction (MLR) assays. Responders (CD4⁺ and CD8⁺ lymphocytes) from the alloantigen and PVCy B6-FoxP3^RFP treatment groups were plated in 96 well flat-bottom plates at 1 × 10⁵ cell/well in triplicate cultured in RPMI media with 2% Fetal Bovine Serum. Responders were co-cultured in the presence of stimulator antigen presenting cells (APC's) irradiated with (20 Gray) from BALB/c (H2^d) or C3H (H2^k) splenocytes at 2 × 10⁵ cells/well. Cultures were incubated for 60 or 132 h. and pulsed with [3H]-thymidine (0.5 Ci/well) for 12 h. Responders were harvested at days 3 and 6 for CPM counts of incorporated [3H]-thymidine isotope measured by liquid scintillation counting (Micro Beta TriLux Counter, Perkin Elmer, Waltham, MA) (30 (link)).

Ex vivo antigen recall proliferation responses were studied using splenocytes and draining inguinal lymph node cells isolated on day 8 after immunization with HuMOG and before onset of disease and under remibrutinib treatment. Isolated cells were counted and equal numbers of cells were added to round-bottom well plates coated either with HuMOG peptide or antiCD3/antiCD28 (R&D Systems, USA and BD, USA, respectively). After 72 h [3H]-thymidine (PerkinElmer, USA) was added and 16 h later cells were harvested and incorporated radioactivity was measured in a MicroBetaTrilux counter (PerkinElmer, Switzerland).