Elispot assay kit

The production of interferon-γ (IFN-γ) from the splenocytes of immunized mice was detected by ELISPOT assay kit (BD Biosciences, USA, San Jose, CA), as described by the manufacturer. Briefly, 96-membrane plates were coated with 0.2 μg of mouse IFN-γ capture antibody and blocked with 10% FBS at 37°C. Splenocytes (1 × 10⁶ cells) in 100 μl of RPMI-1640 medium were applied to each well and stimulated with inactivated influenza pH1N1 virus for an additional 24 hours at 37°C. Plates were then washed and treated with 20 ng of biotinylated mouse IFN-γ detection antibody for 2 hours. Streptavidin-alkaline phosphatase was then added to the wells, and color was developed using an AEC substrate reagent (BD Biosciences). The number of spots was counted using an ELISPOT reader (AID ElispotReader ver.4; AID GmbH, Straßberg, Germany).

The frequencies of IL-2 and IFN-γ secretion by splenocytes were detected by an ELISPOT assay kit (BD) according to the manufacturer’s protocol. Briefly, splenocytes were seeded in 96-well assay plates precoated with 2 μg/mL antibodies against IL-2 and IFN-γ at a final concentration of 3 × 10⁵ cells/well. Recombinant S-trimer 6P protein at 20 µg/mL was used to stimulate S protein-specific T-cell responses, and the same volume of a solution of PMA+ ionomycin was used as a positive control. Spots were counted with an ELISPOT reader system (Autoimmun Diagnostika GmbH, Strassberg, Germany) after immunoimaging [12 (link)].

An ELISPOT assay was performed to analyze IL-2 and IFN-γ production by using an ELISPOT assay kit (BD) according to the manufacturer’s instructions. Briefly, splenocytes from immunized mice were seeded in 96-well plates, and the final concentrations of splenocytes from mice immunized as shown in Table 1 and Table 2 were 5 × 10⁵ cells/well and 2 × 10⁵ cells/well, respectively. The protein gE was used at a final concentration of 20 µg/mL to stimulate gE-specific T-cell responses for 16 h, and the same volumes of medium or PMA + ionomycin were used as the negative control and positive control, respectively. After removal of the cell supernatants, spots were developed by an ALL-IN-ONE mouse ELISPOT Accessory kit and counted with an ELISPOT reader system (Autoimmun Diagnostika GmbH, Strassberg, Germany) [33 (link)].

Spleens were dispersed with a 70 μm cell strainer (NEST, Wuxi, China). After red blood cell lysis with ammonium chloride potassium buffer, splenocytes were collected by centrifugation at 1800 rpm for 5 min, the number of cells was calculated, and the cells were suspended in Roswell Park Memorial Institute (RPMI, Thermo Fisher) 1640 medium supplemented with 10% (v/v) FBS (Biological Industries, Cromwell, CT, USA) and penicillin-streptomycin (Thermo Fisher) at a final concentration of 3 × 10⁶ cells/mL. Then, 100 μL of cells were added to each well of a 96-well plate (Corning Inc., Corning, NY, USA) for further analysis with an ELISPOT assay kit (BD), according to the manufacturer’s protocol. gE and pooled peptides (purity ≥ 95%, synthesized by GL Biochem Co., Ltd. Shanghai, China) at 10 μg/mL were both selected to stimulate gE-specific T cell responses by incubation of cells with protein/peptides overnight. Spots were counted with an ELISPOT reader system (Autoimmun Diagnostika GmbH, Strassberg, Germany) after immunoimaging [26 (link)].

To determine antigen-specific cytokine (IFN-γ and IL-4) responses, splenocytes (5 × 10⁵ cells well⁻¹) were stimulated with Dp antigen (100 μg mL⁻¹) in vitro. After 24 h incubation at 37 °C (95 % room air, 5 % CO2), stimulated splenocytes were washed, and the numbers of IFN-γ- and IL-4-producing cells were determined by using an ELISPOT assay kit (BD Biosciences) according to the manufacturer’s instructions.

For measuring the frequency of antigen-specific CD4⁺ T cells after Mtb infection, single cell suspensions from lungs were prepared and collected in complete IMDM. Lung cells were stimulated for 20 h with mitomycin-D (Sigma-Aldrich)-inactivated spleen cells from uninfected mice that had been pulsed with the MHC class II peptide ESAT-6_1–20 (Research Center Borstel). Detection of antigen-specific IFN-γ-producing CD4⁺ T cells was conducted using ELISPOT assay kits as described by the manufacturer (BD Bioscience and R&D Systems, respectively). Spots were automatically enumerated using an ELISPOT reader (ELISPOT 04 XL; AID) and the frequency of cytokine-producing cells was determined.