Bcip nbt chromogen

ELISpot This assay is designed to determine the proportion of T cells that release IFN-γ after stimulation with SLA in order to quantify the level of stimulation of a specific Th1 polarity immune memory response. It was performed in a manner similar to that previously described [19 (link),27 (link)]. Heparinized blood samples were fractionated by centrifugation over lymphocyte separation medium. The PBMCs obtained were incubated at a density of 10⁶ cells/mL for 3 days in multiscreen HTS filter plates (Millipore, Billerica, USA) previously coated with canine IFN-γ capture antibody (R&D System, Minneapolis, USA), in presence of 10 μg/mL ConA, or 10 μg/mL SLA antigens, or with medium alone, in a humidified 37 °C CO2 incubator. The quantity of IFN-γ was revealed with a specific biotinylated antibody and incubation with Streptavidin-AP and the BCIP/NBT Chromogen (R&D System, Minneapolis, USA). The number of specific spots was determined by an automated ELISpot reader. ConA was used as a positive control and the medium alone was used as a negative control. The data presented are the number of spots per 2 × 10⁵ cells after stimulation with SLA minus the equivalent value obtained with the negative control using medium alone.

Capture antibodies mouse IFN-γ and IL-4 cytokines (3 µg/mL in coating buffer, R&D Systems, USA) were coated on the Multiscreen 96-well filtration plates (Millipore, Billerica, MA, USA), respectively. Spleen cells (n = 3/group) were harvested at 2 weeks post prime-boost immunization. Freshly isolated splenocytes (1 × 10⁶ cells) were cultured on the plate with 100 µL of RPMI 1640 media with 10% FBS, and then stimulated with or without 10 μg/mL of HA-specific peptide (PKGRGLFGAIAGFIENGWEGL) for 36 h in a humidified 37 °C CO₂ incubator. After incubation, the plates were washed with sterile PBS and further incubated with biotinylated anti-mouse IFN-γ and IL-4 antibodies (1:5000 diluted). After three additional washes, alkaline phosphatase (AP) conjugated streptavidin (1: 1000) was added to each well and incubated at room temperature for 3 h. The plates were washed and developed with BCIP/NBT Chromogen (R&D Systems, USA). The number of IFN-γ and IL-4 secreting cells was counted using an ImmunoSpot ELISpot plate reader (Cellular Technology Ltd, Shaker Heights, OH, USA).