Biospot version 5

Mouse IFN-ɣ, IL-4 or IgG ELISPOTs were performed using Mabtech’s ELISPOT BASIC kits (Cincinnati, OH). Briefly, the 96-well Multiscreen plates (EMD Millipore, Billerica, Massachusetts) were pre-coated with 15 µg/ml of mouse-specific anti-IFN-ɣ, anti-IL-4 or anti-IgG antibody, or with 15 µg/ml of H5 rHA. Spleens harvested from H5N1-infected placebo or estradiol-implanted female mice were dissociated and lysed of red blood cells. After washing, resuspended splenocytes were added to anti-mouse IFN-ɣ or IL-4 antibody pre-coated plates at 2.5 × 10⁵ cells/100 µl/well and were incubated with PMA/ionomycin (PMA/IM,1 µg/ml PMA plus 0.75 µg/ml IM) or H5 rHA (10 µg/ml) at 37°C for 40 h. Cells incubated with medium only served as negative controls. For mouse IgG ELISPOT, splenocytes were pre-activated with R848 (1 µg/ml) and mIL-2 (10 ng/ml) before being added to plates coated with anti-mouse IgG or H5 rHA. Spot-forming cells were detected using biotinylated secondary antibodies and counted using the Immunospot Analyzer equipped with Biospot Version 5.0 software (Cellular Technology Ltd, Cleveland, OH). The number of antigen-specific spots were determined by subtracting the number of spots in unstimulated negative control wells and were expressed as number of spots per 10⁶ cells. Each mouse sample was assayed in triplicates.