Donkey anti goat alkaline phosphatase secondary antibody

Spleens from mice after adoptive transfer were isolated and a single cell suspension was prepared. To collect BM cells, tibia and femur were flushed with RPMI media containing 2% FCS and antibiotics. After red blood cell lysis with ACK buffer, cell numbers of splenocytes and BM cells were determined using the Cellometer mini (Nexcelom, USA). 5 × 10⁵ cells were seeded per well on MAIPS Elispot plates (Millipore, MAIPS4510) previously coated with 10 μg/ml Qβ VLPs overnight at 4°C and blocked with 2% BSA in PBS for at least 2 h. After performing a 2-fold dilution series cells were incubated for 5 h at 37°C and 5% CO₂. Subsequently cells were washed off and bound specific antibodies produced by PCs were detected using a goat anti-mouse IgG antibody (EY laboratories, AT-2306-2) followed by a donkey anti-goat alkaline phosphatase secondary antibody (Jackson Immunoresearch, 705-055-147). Spots were visualized by the AP Conjugate Substrate Kit (BioRad, 1706432) and counted using an EliSpot Reader (AID, Germany). The spot size was quantified with the EliSpot 7.0 iSpot software of the EliSpot Reader as the average surface area of the spot.

Spleens from mice after adoptive transfer were isolated and a single cell suspension was prepared. To collect BM cells, tibia and femur were flushed with RPMI media containing 2% FCS and antibiotics. After red blood cell lysis with ACK buffer, cell numbers of splenocytes and BM cells were determined using the Cellometer mini (Nexcelom, USA). 5 × 10⁵ cells were seeded per well on MAIPS ELISPOT plates (Millipore, MAIPS4510) previously coated with 10 μg/ml Qβ VLPs overnight at 4°C and blocked with 2% BSA in PBS for at least 2 h. After performing a 2-fold dilution series, cells were incubated for 5 h at 37°C and 5% CO₂. Subsequently cells were washed off and bound specific antibodies produced by PCs were detected using a goat anti-mouse IgG antibody (EY laboratories, AT-2306-2) followed by a donkey anti-goat alkaline phosphatase secondary antibody (Jackson Immunoresearch, 705-055-147). Spots were visualized by the AP Conjugate Substrate Kit (BioRad, 1706432) and counted using an EliSpot Reader (AID, Germany).