Cytation 5 instrument

The membrane depolarization assessment utilized the cationic dye, 3,3′-dipropylthiadicarbocyanine iodide (DiSC₃(5)), following a modified version of our previously established method [24 (link),55 (link),56 (link),65 (link),91 (link)]. In brief, AB5075 bacterial colonies were cultured overnight on Tryptic Soy agar, and opaque colonies were dispersed in Dulbecco’s Phosphate-Buffered Saline (DPBS, Gibco) until reaching an optical density equivalent to 0.5 on the McFarland standard (~1 × 10⁸ CFU/mL). A bacterial suspension of 4 × 10⁷ CFU/mL was prepared, washed twice with DPBS, and subsequently resuspended in DPBS containing 10 µg/mL DiSC₃(5). A 100 µL aliquot of the bacteria-DiSC₃(5) suspension (n = 3) was dispensed into each well of a black 96-well flat plate (Ultracruz Poly-propylene Microplate sc-204462, Santa Cruz, CA, USA). The plate was incubated at room temperature within a BioTek Cytation 5 instrument and monitored until the fluorescence intensity stopped decreasing. Following this, a 100 µL aliquot of either peptide (final concentration of 50 µg/mL) or DPBS (untreated control) was added into each well, and the plate was promptly returned to the plate reader. Fluorescence readings were captured at 15-s intervals for a duration of 20 min (with excitation at 622 nm and emission at 670 nm). The experiment was conducted twice to ensure reproducibility of results.

ELISA was used for quantification of cytokines in cell-free supernatant and lavage fluid. Cells were centrifuged at 300× g for 5 minutes and the supernatant was collected and stored at −80°C. Murine IL-1α and IL-1β ELISA kits were purchased from R&D Systems. A Biotek Cytation-5 plate reader was used to quantify concentrations.
HEK-blue IL-1R1 cell-line used to measure IL-1α and IL-1β bioactivity was purchased from InvivoGen. For each experiment, 2.8×10⁵ HEK IL-1R1 reporter cells/mL in DMEM (GIBCO) complete media were seeded in 180 μL per well. Twenty μL of each sample was added in duplicates without neutralizing antibodies, with anti-IL-1α neutralizing antibodies (Fisher Scientific), anti-IL-1β neutralizing antibodies (R&D Systems), or both and incubated overnight at 37°C with 5% CO₂. The supernatant was collected and incubated with QUANTI-Blue (InvivoGen) for 30 minutes. SEAP detection and concentration calculations were measured on the Biotek Cytation-5 instrument. IL-1α and IL-1β concentrations were calculated based on a set of standards of known bioactive IL-1α and IL-1β concentrations and presented as pg/mL.

The WM164 cells were cultured for the formation of spheroids in DMEM containing 20 ng/mL epidermal growth factor, 10 ng/mL basal fibroblast growth factor, 5 µg/mL insulin and 0.4% bovine serum. Cells at a concentration of 5 × 10³/mL were added to the medium and incubated with factor B27, growth factor to support the formation of spheroids in cell lines, at a dilution of 1:50 (Gibco, Waltham, MA, USA). The cells were plated onto a 96 well ultralow attachment plate (Costar^®, Corning, NY, USA) 200 µL/well. The wells at the edge of the plate were filled with PBS to ensure adequate humidity inside the plate. After a week of incubation, the resulting spheroids were counted manually and using a Cytation 5 instrument (BioTek, Winooski, VT, USA). Data were analyzed using Gen 5.3 software (BioTek, Winooski, VT, USA).

For immunofluorescence quantification, slides were stained with a primary rabbit polyclonal antibody for IL-17 as above, followed by secondary donkey anti-rabbit IgG antibody conjugated to Alexa Fluor® 594 (Abcam #ab150076) and stored at 4 °C until use. 4′ 6-diamidino-2-phenylindole (DAPI) was used for nuclei staining. Quantification was done by a Biotek Cytation 5 instrument, using the Gen5Image + software (Biotek), which allows for calculation of the percentage of nucleated (DAPI-positive) cells with cytoplasmic IL-17.

Promega CytoTox 96® Non-radioactive Cytotoxicity Assay was used to measure LDH release in cell-free supernatant. Released LDH in culture supernatants was measured with a 30-minute coupled enzymatic assay, which results in conversion of a tetrazolium salt (INT) into a red formazan product. Maximum LDH control was prepared by lysing the same concentration of cells in each experiment with lysis buffer from the Promega kit for 15 minutes prior to collection. LDH read was measured at 490nm with a Biotek Cytation-5 instrument. The % of max LDH release was calculated by dividing the LDH read from each sample by the maximum LDH control read. The maximum LDH control was prepared for each individual experiment.

To test the OX40 receptor blocking activity of Anticalin proteins, NFκB-Luc2/OX40 Jurkat cells were seeded in RPMI1640, 5% FBS at a density of 1 × 10⁴ cells/well in a 384-well plate (Greiner, #781098). Cells were pre-incubated for 30 min at 37°C and 5% CO₂ with titrations of the Anticalin proteins starting at 500 nM in a 3-fold dilution series. In case of co-incubation with serum proteins, 1.5 µM HSA or a mixture of huIgG1, huIgG2 and huIgG4 were added to selected concentrations of Anticalin proteins (0.01 nM, 1.95 nM, 500 nM). NFκB-mediated reporter gene expression was then triggered by stimulation with OX40L for 5 h. A ligand concentration of 3 nM was chosen, resulting in a luciferase activity between 50 and 80% of the maximum signal. Plates were then shortly equilibrated to room temperature before Bio Glo Reagent (Promega, #G7940) was added and incubated for 5 min at RT, followed by readout of luminescence with a Cytation 5 instrument (BioTek).