Duoset development kit

S. enterica serovar Typhimurium strain M525P (49 (link)), a strain of intermediate virulence, was used to establish subclinical infection in vivo. In particular, overnight (stationary phase) bacterial cultures were first washed and resuspended in Dulbecco's PBS (Sigma) and then diluted to the desired dose. WT and Lrrk2^−/− mice were subsequently challenged intravenously with 0.2 ml of the bacterial suspension. The exact dose, as determined by serial dilution and plating the inoculum on LB plates before and after infection, was 1.3 × 10⁴ cfu/mouse. Mice were bled and then euthanized at certain intervals after the initial challenge. Their spleens were aseptically removed and weighed. Mouse serum was analyzed via ELISA for levels of IL-18 (MBL International) and IFN-γ (DuoSet Development Kit, R&D Systems).

IL-1β concentration in cell-culture supernatants was determined using the R&D Systems DuoSet Development kit (DY201, Minneapolis, MN, USA), which detects mature IL-1β (<7% cross-reactivity with pro-IL-1β).

In order to measure the paracrine activity of the MSCs and protein level of BDNF, IL-1β, IL-6 proteins in CM, the supernatant was collected for the quantitative analysis using enzyme-linked immunosorbent assay (ELISA) kits, Human/Mouse BDNF DY248, Human IL-1β/IL-1F2 DY201, and Human IL-6 DY206 with R&D Systems DuoSet Development Kit. Briefly, 100 µL of MSC-CM was added to the ELISA. Plates were coated with a 100 µL monoclonal antibody to the factor of interest, which was incubated for 120 min at 37 °C. Having been washed with PBS, an antibody was added to the wells and the samples were incubated further for 120 min at 37 °C, which were then washed with PBS three times. The substrate solution was added and incubated for 30 min, where the reaction was terminated upon adding the stop solution. The absorbance was recorded at 450 nm using a microplate reader. All ELISA experiments were repeated three times.

Pro-inflammatory and anti-inflammatory biomarkers, including TNF-α (DY510-05), COX-2 (ELK7718), and IL-10 (DY522-05), were analyzed using the R&D Systems DuoSet Development Kit with pre-made ELISA reagent kits as per the instruction of the manual by ELISA (i-mark microplate absorbance reader Bio-Rad (Felipe et al., 2020 (link)).

Detection of IL6 and IL8 was performed by enzyme-linked immunosorbent assay (ELISA), as previously described [21 (link)], using the sandwich method. ELISA plate coating was done using mouse monoclonal anti-human IL8 (clone G265-5, BD Biosciences, San Jose, CA, USA) and IL6 (clone 6708 R&D Systems, Inc.). For detection, biotinylated mouse monoclonal anti-human IL8 (clone G265-8, BD Biosciences) and biotinylated polyclonal goat anti-human IL6 (R&D Systems, Inc.) were utilized with sensitivities of less than 3 pg/mL for IL8 and IL6.
BAL fluids were retrieved before and 48 h after SBP-Ag, concentrated 20-fold and IL1α and IL1β concentrations were determined using a DuoSet Development kit (DY-200 and DY-201, respectively, R&D Systems, Inc.), while EDN concentrations were determined using the sandwich ELISA kit (7630, MBL International Corp., Des Plaines, IL, USA). The IL1α and IL1β DuoSet Development ELISA kits and EDN ELISA kit had sensitivities of 1 pg/mL and 1 ng/mL, respectively.