Ks elispot automated reader

Analysis of B cells for IgG1 production was analyzed as previously described (20 (link)). Briefly, PVDF MultiScreen-IP plates (Millipore, Billerica, MA) were coated overnight (4°C) with anti-IgG1 antibody. 5×10⁵ splenocytes (from naïve or recipient mice) were plated and serial diluted (down to 976 cells/well) with media, incubated overnight (37°C and 5% CO₂), and then lysed with diH₂O. Wells were incubated (4°C, overnight) with alkaline phosphatase conjugated anti-IgG1 (Southern Biotechnology, Birmingham, AL). Plates were developed with BCIP/NBT phosphatase substrate (Sigma), dried, and analyzed by computer-assisted image analysis using a KS ELISPOT Automated Reader using KS ELISPOT software 4.2 (Carl Zeiss Inc, Thornwood, NY). Data is reported as Relative Spot Forming Cells (SFC) per 1×10⁶ splenocytes. A side-by-side ELISA was run in a similar fashion and performed as previously described (21 (link)). Notably, IgG1 standard (Southern Biotechnology) and colormetric analysis (PNPP substrate, Sigma) were utilized to quantitate in vitro antibody production by a Spectramax Plus microplate reader (Molecular Devices, Sunnyvale, CA).

Analysis of B cells for IgG1 production by ELISPOT was performed as previously described (41 (link)). Plates were analyzed by computer-assisted image analysis using a KS ELISPOT Automated Reader with KS ELISPOT software 4.2 (Carl Zeiss Inc, Thornwood, NY). The data are reported as the number of relative Spot Forming Cells (SFC) per 1x10⁶ splenocytes. A side-by-side ELISA was run in a similar fashion and performed as previously described (42 (link)). Colormetric analysis was utilized to quantitate in vitro antibody production by a Spectramax Plus microplate reader (Molecular Devices, Sunnyvale, CA).