Abc ap vector blue substrate reagents

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).

Elispot assays for total IgG were performed as previously described [53]. Briefly, pre-wetted membrane, MultiScreen flat-bottom 96-well ELISpot plates (Millipore) were coated overnight at 4°C with goat anti-human IgG capture Ab (5 μg/mL) or with 2 mg/mL BSA (2% in PBS). To prevent nonspecific binding, plates were then blocked with RPMI 1640 supplemented with 8% FBS for 2 h at 37°C. Subsequently, plates were loaded with cultured blood ASC and were incubated in ~150-200 uL media for ~16–18 h at 37°C in the air incubator (5% CO2). Then cells were removed and the plates were washed six times with washing buffer using Microplate Washer (Biotek). Secondary goat anti-human IgG alkaline phosphatase-conjugated Ab (1 μg/mL, diluted in PBST + 2% BSA), was then added and incubated for ~2 h at RT. Spots were developed and visualized with an enzymatic color reaction using ABC-AP Vector Blue Substrate reagents (Vector Laboratories). Plates were counted on the ELISpot reader (Cellular Technology Limited; CTL) using the ImmunoSpot 5.0.9.21 software.