Spectramax 340pc microplate reader

Serum EPO levels were measured using the Mouse EPO Quantikine^® ELISA kit according to manufacturer’s instructions. Briefly, serum samples and EPO standards were diluted two-fold and 50 μL of each sample was added in duplicate to the appropriate wells of a microplate pre-coated with an EPO specific monoclonal antibody. The plate was then covered and incubated at RT for 2 h on a microplate shaker. Following incubation, the plate was washed five times. After the last wash, the plate was emptied and 100 μL of mouse EPO monoclonal antibody conjugated to horse radish peroxidase was added to each well and incubated at RT for 2 h on a microplate shaker. The plate was washed again and 100 μL of substrate solution (hydrogen peroxide and tetramethylbenzidine) was added to each well and incubated at RT for 30 mins. Colorimetric reactions were stopped by adding 100 μL of 0.25 N hydrochloric acid to each well and the absorbance was read immediately at 450 nm and 540 nm using a SpectraMax® 340PC microplate reader (Molecular Devices, Sunnyvale, CA). Readings at 540 nm were subtracted from those at 450 nm to correct for optical imperfections in the plate and the corrected values were used for subsequent analysis. Sample concentrations were determined using a four-parameter logistic standard curve.

Soluble ICOSL concentrations were determined using the Human B7-H2 Duo Kit ELISA (R&D #DY165–05) and the DuoSet ELISA Ancillary Reagent Kit 2 (R&D #DY008). R&D company protocols were followed. Cell culture supernatants were diluted at either a 1:5 ratio (AdV/DC supernatants) or 1:2 ratio (mDC supernatants). All standards and samples were run in triplicate. Absorbance readings were read using the Spectramax 340PC Microplate Reader (Molecular Devices). The concentrations of the standard controls were used to generate a standard curve using SoftMax Pro Software (Version 4). Sample concentrations were determined by interpolating absorbance readings of samples to the generated standard curve.

For flow cytometric crossmatch, BALB/c splenocytes were processed into single-cell suspensions and pre-treated with Fc Block (BioLegend), followed by incubation with recipient serum at 4°C. Splenocytes were then washed and labeled with surface markers and anti-mouse IgG (BioLegend) for quantification of anti-BALB/c IgG by flow cytometry. For ELISA anti-OVA antibody measurements, flat-bottom 96-well Immulon 4HBX microtiter plates (VWR) were coated with OVA protein (100 μg/well; Sigma-Aldrich) overnight at 4°C, blocked with 10% FBS in PBS-T for 1 hour at 37°C, and then incubated with serum samples for 1.5 hours at 37°C. OVA-specific immunoglobulins were detected with HRP goat anti-mouse IgG (Poly4053, BioLegend), developed by using the TMB substrate system (Thermo Scientific), and read at 450 nm on a Spectra MAX 340PC Microplate reader (Molecular Devices).