Varioskan flash luminometer

Competence development was measured using a modified version of the method of Caymaris et al. [24 (link)]. Stock cultures were grown at 37°C in TSB pH 8 to OD_600nm of 0.2 and frozen with 15% glycerol at −80°C. Thawed cultures were used to inoculate (1 : 10) C + Y medium containing luciferin (0.18 mg ml⁻¹). Three hundred microlitres per well was added to a 96-well plate. To determine the synergistic effect of AliB-like ORF 1 ligand (SETTFGRDFN) on CSP 2-mediated competence induction, the bacteria were pre-treated for 30 min with AliB-like ORF 1 ligand (100 μg ml⁻¹) before addition of 100 ng ml⁻¹ CSP 2. To measure competence, we used ssbB::luc transcriptional fusion (kindly provided by M. Prudhomme and J. P. Claverys, Centre National de la Recherche Scientifique, Toulouse, France), which reports competence through light emission by luciferase [7 (link)]. Relative luminescence unit and OD values were recorded throughout incubation at 37°C (in a Varioskan Flash luminometer; Thermo Fisher Scientific, Zurich, Switzerland). Area under the curve (AUC) was calculated using GraphPad Prism 5.

SW872 cells were cultured in 24well plates (DMEM 4.5 g glucose, 10% FBS, 1%P/S) for 24 h prior to transfection. 500 ng plasmid DNA was transfected (six replicates per transfection). pGL CMV Renilla (Promega UK) was co-transfected as a control. Transfections were carried out using FuGENE HD (Switchgear Genomics USA) following manufacturers' guidelines. Transfected cells were cultured for 48 h prior to harvesting. Luciferase assays were carried out using the Dual-Luciferase® Reporter Assay System (Promega UK), on a VarioSkan Flash Luminometer (ThermoScientific). For estrogen treatment, cells were cultured in phenol-free media and charcol-stripped FBS for 48 h prior to addition of 10 nM E2 (Sigma E2758). Cells were cultured for 72 h then harvested for RNA.

BHK21 cells transfected with WT or mutant replicon plasmids were lysed using cell lysis buffer (Promega, USA) and harvested at various time points. A Renillaluciferase assay system (Promega) and a Varioskan Flash luminometer (Thermo Scientific, USA) were used to detect the Rluc activity according to the manufacturer's instructions.

Mice were sacrificed and draining popliteal lymph nodes and tibialis anterior muscles excised. Lymph nodes were washed in PBS, crushed and cells were recovered by passage through a 70 μm strainer. Cells were lysed in NP-40 lysis buffer to release luciferase for ex vivo detection. Tibialis anterior muscles were macerated in NP-40 lysis buffer with glass beads using a Mini Beadbeater (Biospec Products) at maximum speed (4800 oscillations min⁻¹). Homogenised material was centrifuged at 4000 rpm for 4 min, and supernatant collected. Supernatants from lymph nodes and muscle extracts were assayed for Photinus luciferase activity using the BrightGlow^TM Luciferase Assay System (Promega, UK) according to manufacturers instructions. Luminescence was measured using a Varioskan Flash luminometer (Thermo Scientific).

One day prior to performing the assay, GripTite 293 cells (Invitrogen) were seeded in 96-well plates (3 × 10⁴ cells/well). Heat inactivated test samples were diluted 4-fold from 1:9 to 1:2,304 in 10% FBS in DMEM and incubated 1:1 with ChAd63 expressing the secreted alkaline phosphatase gene (8 × 10⁷ vp/mL) for 1 hr at 37°C. Serum and virus were then added to 293 cells in a volume of 200 μL in duplicate for 1 hr, after which sample and virus were aspirated and replaced with fresh 10% FB DMEM. A virus-only control was included. After 22–26 hr at 37°C, 50 μL of medium was assayed for SEAP activity using a Phospha-Light TROPIX phosphatase assay (Applied Biosystems) in black assay plates, and luminescence was measured after 45 min on a Thermo-Fisher Varioskan Flash Luminometer. Anti-vector neutralization titers were defined as the dilution of serum showing 50% reduction in SEAP activity, based on observed % inhibition values relative to SEAP activity from virus alone. For trial A, anti-vector antibodies were measured in serum and for trial B plasma was used, however, we have determined that these sample types are equivalent for this assay.

Antibody responses to TRAP were measured using a luciferase immunoprecipitation system (LIPS) as previously described⁵⁰ . The assay is based on binding of immobilised antibodies to a fusion protein of TRAP (P. falciparum 3D7 sequence) (human Ii or macaque Ii) and Renilla luciferase (rLuc). Briefly, serum samples were incubated for 1 hour with a cell lysate from 293 cells transfected with a TRAP-rLuc (hCD74-rLuc or macCD74-rLuc) expression plasmid, prior to incubation with Protein A/G UltraLink Resin beads (Thermo-Scientific) in MultiScreen HTS membrane Barex plates (Millipore) for 1 hour. Unbound lysate and antibodies were removed by washing the plates prior to quantification of bound rLuc activity using Renilla luciferase assay system (Promega) and a Varioskan Flash luminometer (Thermo). Antibody levels are expressed as log10 luminescence units.