Victor3 multilabel plate reader

For anti-CD83 ELISAs, plates were coated overnight with 1 µg/ml mouse CD83-Fc (Sino Biologicals) and blocked with 5% BSA. Plates were overlayed with rat anti-mouse CD83 antibody, 1:100–1:1,000 diluted serum or hybridoma supernatant (50 µl) and then an HRP conjugated goat anti-rat IgG Fc-specific antibody (Sigma-Aldrich). The HRP substrate SIGMAFAST OPD solution (Sigma-Aldrich) was used to detect binding of antibodies by reading absorbance at 450 nm on a Victor3 Multilabel Plate Reader (Perkin Elmer). Wells were washed with 0.05% Tween 20 in PBS between steps. To detect anti-DCR-5 antibodies, ELISA plates were coated with 10 µg/ml DCR-5 and blocked with 5% BSA. Plates were overlayed with serum samples diluted 1:10,000 and 1:100,000 in PBS, without incubation (no spike) or after incubation for 30mins with 10µg/ml DCR-5 (spike). Bound mouse antibodies were detected with HRP conjugated pre-adsorbed goat anti-mouse IgG (ab97040, Abcam, Cambridge, MA), measured as above. Anti-collagen antibodies produced in the CIA model were measured in 1:500 and 1:40,000 diluted serums using the mouse anti-type I/II collagen IgG assay kit with TMB (Chondrex, Redmond WA) under manufacturers’ instructions. The mouse IL-10 ELISA (ThermoFisher) performed on FL-DC supernatants was conducted using the manufacturer’s instructions.

For fluorescence reading, 250 µl of bacteria and isolated bacterial lysates in Tris buffer (50 mM, pH 7.4) were prepared in 96-well black microplates (Greiner). 2.5 µl of probe 1 (1 mM) was added to test wells, and the same volume of DMSO (1 mM) was added to control wells. The fluorescence intensity (λ_ex = 327 nm, λ_em = 415 nm) was measured at 37 °C in a time-dependent manner by the Victor3 multilabel plate reader (Perkin-Elmer) (0-240 min). For precipitation observations, 1 mM probe 1 with bacteria or bacterial lysates in Tris buffer (50 mM pH 7.4) were prepared in the 96-well white plate (Falcon) and incubated at 37 °C for 24 h.

Vegetative cells were diluted to approximately 2.5 × 10⁵ CFU ml⁻¹ in fresh Dulbecco’s modified Eagle’s medium (Gibco-Invitrogen, Carlsbad, CA) containing 10% (vol/vol) fetal bovine serum (HyClone, Logan, UT) and the various concentrations of CXCL10 or CTTC to be tested. Aliquots of 100 µl were placed in triplicate wells of a 96-well plate and incubated at 37°C with 5% CO₂ for approximately 4 h, unless otherwise noted, at which point alamarBlue dye (AbD Serotech, Oxford, United Kingdom) was added at a 1:10 dilution and allowed to reach visual saturation in an untreated control under protection from light. Active metabolism of the vegetative cells was quantified via the generation of a fluorescent signal from the reduction of the alamarBlue oxidation-reduction dye by measuring the fluorescence at 530-nm excitation and 590-nm emission wavelengths with a PerkinElmer Victor³ multilabel plate reader (PerkinElmer, Waltham, MA). The percentage of the control was calculated by comparison of the reading obtained with the chemokine-treated samples to that obtained with the corresponding untreated samples. Light microscopy was used for bacterial cell visualization. Camera control and image capture were performed with the QCapture pro-5.1 software as previously described (17 (link), 35 (link)).

In vitro translation assay was performed as described in [ref. ^{40 (link)}]. Briefly, translation of luciferase mRNA (Promega) was performed with nuclease-treated rabbit reticulocyte lysate (Promega). Each reaction mix (30 µl) contained the following components: 12.6 µl of rabbit reticulocyte lysate, 0.5 µl of Luciferase mRNA (1 mg/ml), 0.3 μl of amino acid mixture minus leucine (1 mM), 0.3 μl of amino acid mixture minus methionine (1 mM), and 16.3 μl of proteins, puromycin (Sigma) or buffer (25 mM NaPO4 pH 7.4, 50 mM NaCl, 2 mM DTT). Reactions were assembled at different concentration of RGG or RGG-Ub (20-10-5-2.5-1.2-0.6-0.3 µM) or BSA (10 µM). All in vitro translation reactions were prepared on ice, followed by incubation at room temperature for 3 h before luminescence measurement. Luciferase translation was measured with luciferase assay system (Promega) by mixing 75 µl of luciferase substrate with 3 µl of unpurified translation mixture in a black 96-well plate (Corning). End-point luminescence measurements were carried out in triplicate using Victor3 multilabel plate reader (Perkin Elmer).

For the description of cell proliferation, we used the MTT cell proliferation assay kit (Invitrogen). The cells from each population in P3 were plated in 96-well plates, seeding density 1 × 10⁴ cell/well and cultivated in standard cultivation medium (DMEM-F12 w/o phenol red + 10% FBS + 2% ATB + ATM) at 37 °C and 5% CO₂ for 24, 48, 168 and 240 h. After the cultivation period, we removed the medium and replaced it with 100 µL of fresh culture medium. In the next step, 10 µL of 12 mM MTT stock solution to each well was added and incubated at 37 °C for 4 h. At the end of incubation time, we added 100 µL of SDS-HCl solution, mixed it and incubated. After 12 h, the content of each well was mixed carefully by the pipette and the absorbance was measured at 572 nm by Perkin Elmer Victor3 Multilabel Plate Reader. Statistical analyses were processed via two-way ANOVA, followed by the Tukey test, with the mean considered from five measurements.