Multiskan fc multiplate photometer

Human HeLa cells were used in the assay. Ninety μL of 1 × 10⁵ mL⁻¹ cells were placed into the wells of 96-microwell plates. After 12 h, 10 μL of a medium containing test compounds at various concentrations were added to the cells which were then incubated at 37 °C for 48 h. Afterward, the supernatant was removed and 20 μL of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (2.5 mg mL⁻¹) in PBS were added to each well. After incubation at 37 °C for 3 h, 80 μL of dimethyl sulfoxide (DMSO) were added to each well and incubated for an additional 15 min. The absorbance was then measured at 570 nm with a Thermo Scientific Multiskan FC multiplate photometer.
Hemolytic activity was determined with red blood cells freshly isolated from healthy rabbits. Brevicidine and laterocidine were added at final concentrations of 128, 64, 32, and 16 μg mL⁻¹ in 0.5% DMSO to 2% (v/v) erythrocytes in PBS. The cells were incubated for 1 h at 37 °C and centrifuged for 5 min at 10,000 × g. The supernatant was transferred to a 96-well plate and the absorbance was measured at a wavelength of 570 nm with a Thermo Scientific Multiskan FC multiplate photometer. The absorbance relative to the positive control, which was treated with 10% Triton X-100, was defined as the percentage of hemolysis.

Dex-FITC in the sera was measured using a custom-built ELISA. Ninety-six well plates were coated with anti-FITC monoclonal antibody (1F8-1E4, Invitrogen) at 4°C overnight and blocked with 1% bovine serum albumin (BSA), 0.05% Tween 20, PBS. Mouse sera were 1/10 diluted with PBS and added to the plate and incubated at room temperature for 2 h. After washing with PBST, the plate was incubated with a biotin-conjugated anti-FITC monoclonal antibody (NAWESLEE, eBioscience) for 1 h, followed by streptavidin-horseradish peroxidase (R&D systems) for 20 min. TMB substrate reagent (R&D systems) was then added to each well and the plate was incubated for a further 30 min; 2N H₂SO₄ solution was added to stop the reaction. The absorbance at 450 nm was measured using a Multiskan FC multiplate photometer (Thermo Fisher Scientific), and the basal absorbance at 570 nm was subtracted from the values.

IL-13 concentrations were measured with an ELISA Development Kit (Peprotech). IL-4 ELISA were performed with α -IL-4 (clone 11B11, BioXcell) as coating antibody and biotinylated α-IL-4 (clone BVD6-24G2, Southern Biotech, Birmingham, AL) as detection Ab. Alkaline phosphatase-coupled streptavidin and para-Nitrophenylphosphat substrate (both from Southern Biotec) were used for detection. GM-CSF concentrations were measured with a commercial ELISA kit (R&D Systems). ELISAs were measured with a Multiskan FC multiplate photometer (ThermoScientific, Waltham, MA).