Automatic elispot reader

IFN-γ and IL-2-based ELISPOT assays were performed to detect antigen-specific T lymphocyte responses [10 (link)]. Spleens of C57BL/6 mice were removed after one week of booster immunization, and splenocytes were isolated. 96-well flat-bottom plates were pre-coated with 10 μg/ml of anti-mouse IFN-γ Ab or anti-mouse IL-2 Ab (BD Biosciences, USA) and stored at 4°C overnight. RPMI 1640 containing 10% FBS was added with 200 μl/well and incubated for 2 h at room temperature to block the wells. Then, 0.1 MOI of VACV was diluted in RPMI 1640 containing 10% FBS and added 100 μl/well to ELISPOT plate microwells. Meanwhile, the cell suspension was prepared at 1 × 10⁷ cells/ml, and 100 μl/well of cell suspension was added to each well which was incubated in a 37°C, 5% CO₂ incubator. After 24 h of incubation, the cells were removed, and the plates were processed in turn with biotinylated IFN-γ or IL-2 detection antibody (BD Biosciences, USA), streptavidin-HRP conjugate (BD Biosciences, USA), and AEC substrate (BD Biosciences, USA). After waiting 15 min for visible spots, the plates were washed clean with deionized water and dried. The number of spots was counted, and the ELISPOT plate microwells were photographed using an automatic ELISPOT reader and image analysis software (Cellular Technology Ltd.).

To detect antigen-specific T lymphocyte responses, an IFNγ-based ELISpot assay was performed as previously described [46 (link)], with some modifications. Briefly, an S peptide pool consisting of 15–18-mers (overlapping by 11 amino acids) and spanning the entire S protein of SARS-CoV-2 were synthesized. Spleens of vaccinated BALB/c mice were harvested at 2 weeks post the second dose immunization and plenocytes were isolated. Flat-bottom, 96-well plates were precoated with 10 μg/mL anti-mouse IFNγ Ab (BD Biosciences, USA) overnight at 4°C, and then blocked for 2 h at 37°C. Mouse splenocytes were added to the plate. Then, the peptide pool (2 μg /ml individual peptide) was added to the wells. Phytohemagglutinin (PHA) was added as a positive control. Cells incubated without stimulation were employed as a negative control. After 24 h of incubation, the cells were removed, and the plates were processed in turn with biotinylated IFNγ detection antibody, streptavidin-HRP conjugate, and substrate. When the coloured spots were intense enough to be visually observed, the development was stopped by thoroughly rinsing samples with deionized water. The numbers of the spots were determined using an automatic ELISpot reader and image analysis software (Cellular Technology Ltd.).

TCR-T cell responses were detected by IFN-γ-specific enzyme-linked immunospot assay (ELISPOT) (BD Bioscience, cat. 551849) and ELISA (BD Bioscience, cat. 555142) as previously described^{30 (link),46 (link)}. In brief, 96-well ELISPOT plates were coated overnight at 4 °C with anti-human IFN-γ antibody dilutions. TCR-T cells as effector cells (1 × 10⁵ cells per well) and different tumor cells as target cells (5 × 10⁴ cells per well) were mixed in wells and incubated for 18 h, the number refered in this study was the total number of T cells. PMA/ION was used as a positive control for non-specific stimulation, and cells incubated without stimulation were used as a negative control. After incubation, cells were removed and the plates were processed according to the manufacturer’s instructions. The number of spots was captured and quantified using an automatic ELISPOT reader and image analysis software (Cellular Technology Limited). After peptides immunization, the peptide-specific T cells in the mouse spleen were also determined using the IFN-γ-specific ELISPOT assay (BD Bioscience, cat. 551881).

The CTL epitope-specific response was measured by performing IFN-γ ELISPOT assays as described previously [17 (link)]. Previously identified HLA class I-restricted epitopes (8–11 residues) of influenza virus were retrieved from published data [17 (link), 18 (link)]. Conserved peptides between H7N9 and 2009pH1N1 were mixed into a conserved peptide pool as described in reference (JID reference) [19 ]. Mutated HLA class I-restricted peptides between H7N9 and 2009pH1N1 were considered as H7N9-specific peptide pools (JID reference). The number of spots was determined using an automatic ELISPOT reader and image analysis software (Cellular Technology Limited).

IFN-γ and IL-2-based ELISPOT assays were performed to detect antigen-specific T lymphocyte responses [10 (link)]. Spleens of C57BL/6 mice were removed after one week of booster immunization, and splenocytes were isolated. 96-well flat-bottom plates were pre-coated with 10 μg/ml of anti-mouse IFN-γ Ab or anti-mouse IL-2 Ab (BD Biosciences, USA) and stored at 4°C overnight. RPMI 1640 containing 10% FBS was added with 200 μl/well and incubated for 2 h at room temperature to block the wells. Then, 0.1 MOI of VACV was diluted in RPMI 1640 containing 10% FBS and added 100 μl/well to ELISPOT plate microwells. Meanwhile, the cell suspension was prepared at 1 × 10⁷ cells/ml, and 100 μl/well of cell suspension was added to each well which was incubated in a 37°C, 5% CO₂ incubator. After 24 h of incubation, the cells were removed, and the plates were processed in turn with biotinylated IFN-γ or IL-2 detection antibody (BD Biosciences, USA), streptavidin-HRP conjugate (BD Biosciences, USA), and AEC substrate (BD Biosciences, USA). After waiting 15 min for visible spots, the plates were washed clean with deionized water and dried. The number of spots was counted, and the ELISPOT plate microwells were photographed using an automatic ELISPOT reader and image analysis software (Cellular Technology Ltd.).