Quantifying IgG-Secreting Cells via ELISpot

To assess survival and Ig secretion function of cultured ASC, enzyme-linked immunospot (ELISpot) assay was used to quantify IgG-secreting cells in the cultures. These ELISpot assays used goat anti-human IgG for coating and alkaline phosphatase-conjugated goat anti-human IgG for detection^{36 (link),49 (link)}. Briefly, pre-wetted ELISpot plates (Millipore) were coated overnight at 4 °C with goat anti-human IgG (H + L) capture antibody (Novex/Fisher) or with BSA. Plates were then blocked with RPMI 1640 supplemented with 8% FBS and subsequently loaded with cultured ASCs and incubated at 37 °C for 16–18 h. Cells were removed and plates were washed six times. Bound Abs were detected using alkaline phosphatase-conjugated goat anti-human IgG secondary antibody (Jackson Immunoresearch). Spots were developed with ABC-AP Vector Blue Substrate reagents (Vector Laboratories). The spots were quantified using the Cellular Technology Limited; CTL (ImmunoSpot 5.0.9.21 software).