Mithras2 lb 943 monochromator multimode reader

Following cell-free protein synthesis in the presence of ¹⁴C-leucine, reactions were fractionated into the supernatant and the microsomal fraction by centrifugation at 16.000 × g and 4 °C for 10 minutes. The microsomal fractions were resuspended in equal volumes of PBS. Subsequently, aliquots of 5 μl (from templates without IRES) or 2.5 μl (from templates with IRES) were subjected to hot trichloroacetic acid precipitation and liquid scintillation counting in triplicates as described previously17 (link). Total protein yields were calculated from measured disintegrations per minute (dpm) taking into account the specific radioactivity, the molecular mass and the number of leucines of the synthesized proteins.
Fluorescence of the fusion proteins was measured from 5 μl aliquots of the corresponding fractions in 95 μl PBS solution on black 96 well microplates (Berthold) using the “Mithras2 LB 943 Monochromator Multimode Reader” (Berthold). For eYFP detection, samples were excited at 485 nm and emission was detected at 530 nm. For mCherry detection samples were excited at 540 nm and emission was detected at 590 nm. The background fluorescence of control measurements from cell-free reactions carried out under identical conditions but in the absence of a gene template was subtracted from the measured intensities.

Influenza A H1N1 (A/SOLOMON ISLANDS/3/2006) (Interchim, Montluçon, France) was coated on Maxisorp 96-well plates (2 µg/mL in PBS-1% milk) overnight at 4 °C. After 3 washes in PBS-0.01% Tween-20, the Nbs were added (1 µM in PBS milk) for 1 h. Plates were washed thrice and incubated with HRP-coupled anti-c-myc 9E10 antibody (Abcam, Cambridge, MA, USA) diluted at 1:2000 in PBS milk. After three final washes, the luminescence signal (Pierce ECL Western Blotting Substrate, ThermoFischer Scientific, Waltham, MA, USA) was read in a multiplate reader (Mithras² LB 943 Monochromator Multimode Reader, Berthold Technologies, Thoiry, France).

The CXCR4-Rluc8, the YPET-β arrestin 2, and the CAMYEL constructions were provided by Dr. Mohammed Akli Ayoub (Al Ain University, United Arab Emirates). HEK293FT cells were transfected in 96-well plates with 50 ng of plasmid DNA and 0.5 µL of Metafectene (Biontex Laboratories GmbH, München, Germany) per well, according to the manufacturer instructions. Forty-eight hours after transfection, Nbs were added to the cells in 10 mM PBS—1 mM Hepes and incubated for 2 h at 37 °C. Recombinant SDF1-α (Peprotech, Rocky Hill, NJ, USA) was added for 15 min at a final concentration of 50 nM. Coelenterazin H (Interchim, Montluçon, France) at a final concentration of 50 µM was added to the medium right before the measurement. Forskolin (Sigma Aldrich-Merck, Merck KGaA, Darmstadt, Germany) at a final concentration 100 µM was mixed to Coelenterazin H in CAMYEL assays. Plates were read on a Mithras² LB 943 Monochromator Multimode Reader (Berthold Technologies, Thoiry, France).