Elisa reader

Migration assays were performed using 24-well Transwell^® culture chambers (Costar, Cambridge, MA). Lower chambers were filled with Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Equal numbers (3×10⁴) of SW982 human synovial fibroblasts were added to the upper insert in serum-free DMEM. Transwell chambers were incubated at 37°C in a 5% CO₂ humidified incubator for 24 h. After incubation, cells that had migrated to the underside of the membrane were fixed in methanol and stained with crystal violet. After imaging, stain was eluted from the cells in 10% acetic acid, and optical density (O.D.) at 570 nm was measured using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA). The migratory ability of cells was assessed in triplicate wells.

Shigella-specific protein, IpaB, IpaC (Venkatesan et al., 1988 (link)), and IcsP (Fukuda et al., 1995 (link)), and Shigella whole cell-specific antibody levels in blood serum and BAL fluid were measured by ELISA as described previously (Shere et al., 1997 (link); Kim et al., 2015 (link)). Briefly, 96 well-plates (Nunc., Rockilde, Denmark), were coated with 200 ng/well of IpaB, IpaC, PSSP-1, LPS (S. flexneri 2a) in 100 μl of PBS, at 4°C overnight. For whole-cell coating, 100 μl of 5 × 10⁵ cells/well of F.I.-Shigella whole cells in PBS were incubated for 4 h at RT followed by overnight at 4°C. After blocking with blocking buffer (1% BSA in PBS), serial dilutions of sera or BAL fluids in blocking buffer were incubated for 2 h at RT. Then, HRP conjugated goat anti-mouse IgG (1:5,000, Southern Biotech) were incubated for 1 h at RT. After final washing, peroxidase substrate (TMB; Moss, Pasadena, MD) was added per well for 10–15 min and 0.5 N HCl was added for stopping the reaction. The OD was measured in an ELISA reader (Molecular Devices, Sunnyvale, CA). The antibody titer was expressed as the reciprocal log2 titer of dilution showing 0.2 of absorbance at 450 nm.

Whole-cell antimicrobial activities were determined using a broth microdilution method described previously [10 (link)]. Most of the test strains were grown to mid-log phase in Mueller–Hinton broth and diluted 1,000-fold in the same medium. Cells (10⁵/mL) were dispensed at 0.2 mL/well in 96-well microtiter plates. Streptococcus pneumonia and Acinetobacter baumanii were grown in Todd–Hewitt medium and nutrient broth, respectively. The test compounds and ciprofloxacin (Sigma St. Louis, MO, USA) were soluble in DMSO, the final concentration of which did not exceed 0.05% in the cells. Cells were treated with 0.05% DMSO as a vehicle control. The MICs were determined in triplicate by serial two-fold dilutions of the test compounds. The MIC was defined as the concentration of a test compound that completely inhibited cell growth during a 24-h incubation period at 37 °C. Bacterial growth was determined by measuring the absorption at 650 nm using a microtiter enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices Corporation, Sunnyvale, CA, USA).

Analysis of plasma cytokine levels was performed using the Beadlyte^® Mouse Multi-Cytokine Detection System (Upstate, Charles, MO, USA) and the Luminex100 luminometer (Luminex Corporation, Austin, TX, USA) according to the manufacturer’s instructions. Fifty-microliter aliquots of plasma were loaded into 96-well microplates with 25 μL of Luminex bead-conjugated anti-mouse multi-cytokine antibody. After 2 h, 25 μL of biotin-conjugated anti-mouse multi-cytokine antibody was added to each well. After incubation for 1.5 h, 25 μL of the streptavidin-phycoerythrin solution was added. After 30 min, 25 μL of stop solution was added. Samples were analyzed using a Luminex 100 luminometer (Luminex, Austin, TX, USA). Quantification of cytokines was performed by regression analysis from a standard curve generated from cytokine standards included in the kit with a lower limit of detection of 10 pg/mL for all cytokines evaluated.
Caudal DRG or spinal cord tissues were homogenized in lysis buffer, and centrifuged at 14,000 rpm (19,300× g) for 20 min at 4 °C. The resulting supernatants were then used for further analysis. IL-1β secretion was quantified using ELISA kits (R&D system, Minneapolis, MN, USA) according to the manufacturer’s instructions. The absorbance of the reaction was read on 450 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

We used the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) to evaluate cell viability. CD8⁺ T cells (1 × 10⁵/well) were cultured in 96-well plates and treated for 48 h with Tim-3 and/or PD-1 antibody as described earlier. Thereafter, 10 μl CCK-8 was added into each well and incubated at 37 °C for 0.5–4 h. The optical density at 450 nm was determined using an enzyme-linked immunosorbent assay (ELISA) Reader (Molecular Devices, Sunnyvale, CA, USA) to measure cell viability.

At day 7 and 21 post-transduction, HMCs were seeded onto a 96-well plate at a concentration of 500 cells/well. Non-transduced HMCs were seeded at the same concentration, as a control. 10 μl of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, Sigma) diluted in ddH₂O (5 mg/ml) was added to each well, and the plate was incubated at 37 °C for 4 hours. Following incubation, 100 μl of dimethyl sulphoxide (DMSO, Sigma) was added to each well. Optical Density (OD) was read at 570 nm and 650 nm with an ELISA reader (Molecular Devices). In one set of experiments cells were treated at the time of the seeding with 5 μM DZnep.
To trigger apoptosis, HMCs were incubated with 50 μM of etoposide (Merck) for 24 h as follow. At day 7 post transduction miR302, scramble and non-transduced cells were seeded at confluency of 50%. The day after the cells were gently washed with PBS (Sigma) and fresh media was added along with 50 μM of etoposide. The cells were lysed in RIPA buffer after 24 h.