Genios pro reader

The levels of female sex hormones (FSH, E2, LH and PGN ) were measured in the saliva samples, in the different experimental conditions, by SinglePlex ELISA kit with a GENios-Pro Reader (Tecan) following the manufacturer's instructions.

The effect of mouse sera and rPfRH5 on the parasite invasion of human red blood cells (RBCs) was assessed in three experiments using an invasion inhibition assay. Synchronized trophozoites of the P. falciparum CY and 3D7 strains were purified by centrifugation at 1,500 × g and 4°C for 15 min on Percoll gradients (16 (link)). To obtain a final hematocrit of 2% and parasitemia of 0.2%, ~7.2×10⁵ schizonts in 180 ml complete medium with 10% human serum were mixed with 4×10⁷ human RBCs. Each well received 180 μl of the culture and 20 μl heat-inactivated (56°C, 30 min) mouse anti-PfRH5 sera (or rPfRH5 protein). The medium was changed every 24 h. After incubation for 72 h, the number of parasites was determined; the parasites were collected by centrifugation (3 min at 1,000 × g) and the supernatant was removed by aspiration (17 (link)). Hoechst 33258 stain (no. 861405; Sigma-Aldrich) in PBS (0.1 ml) was added to each well to cover the complete surface of the well and left for 10 min at 37°C. The stain was removed by centrifugation and the RBCs were washed twice with PBS. Fluorescence intensity was read at 450 nm after 10 min using a GENios Pro Reader (Tecan, Männedorf, Switzerland). The percent inhibition was calculated using the fluorescence intensity data in the pre-immunization serum controls as 100% invasion.

Biotinylated N-Avitagged-TEAD4^217–434 (1 nM) and LANCE Eu-W1024 Streptavidin (0.5 nM, PerkinElmer) were pre-incubated for 1 h at room temperature in 50 mM HEPES pH 7.4, 100 mM KCl, 0.05% (v/v) Tween-20, 0.25 mM TCEP, 1 mM, and 0.05% (w/v) BSA. Different YAP mutants (20 nM) and serial dilutions of them were added and incubated in white 384-well plates (Greiner Bio-One International) for 1 h at room temperature. DMSO was present at 2% in the assay. The solubility of the peptides in assay buffer was measured by dynamic light scattering with a Dyna Protdevice (Wyatt technology Corp.). The fluorescence in the TR- FRET assay was measured with a Genios Pro reader (Tecan) (50 μs delay between excitation and fluores- cence, 75 μs integration time, excitation wavelength 340 nm, emission wavelengths 620 and 665 nm). Data analyses were carried out using the TR-FRET 665/620 nm emission ratio. The IC₅₀ values were obtained by nonlinear regression analysis with GraphPad Prism (GraphPad Software) (Bokhovchuk et al., 2020a (link)).

Intestinal permeability was measured as described previously (8 (link)): After 6 days, mice were anaesthetized mice using isoflurane (Forene, Abbott, Wiesbaden, Germany). Following laparotomy, the colon was mobilized and opened at the ileocaecal valve and at the upper rectum. After flushing the colon with PBS at RT to remove blood and stool, the colon was ligated at the cutted ends without compromising the blood supply. To determine intestinal permeability 200µl of 4kDa FITC dextran diluted in PBS (1 mg/ml) was injected in the ligated colon. After 1h blood from the inferior vena cava was taken to measure the concentration of 4 kDa FITC dextran translocated from the colonic lumen into the blood. The blood samples were centrifuged at 13.400 rpm for 10 minutes at 4°C, and the luminescence of the serum was quantified by using Genios Pro Reader (Tecan, Maennedorf, Switzerland). After blood collection mice were euthanized by exsanguination, the colon was harvested and the length was measured.
The ligations of the colon were removed and the colon was cut longitudinally into two pieces. One part was fixed in 4% paraformaldehyde embedded in paraffin and sectioned. The other half of the colon was lyzed and homogenized with a Tissue Lyzer (Quiagen, Hilden, Germany) in a SDS lysis buffer and used for Western Blot analysis.