Immunospot s6 analyzer

For ELISA, serum was prepared from the blood of μMT chimeric mice. ELISA plates (Nunc Maxisorp) were coated with NP20-BSA (Biosearch Technologies) and blocked with 1% BSA in PBS. Serial dilutions of serum samples (0.1% BSA/PBS) were added and incubated overnight. NP-specific IgM and IgG3 antibodies were detected using biotinylated anti-mouse IgM- or IgG3-specific immunoglobulins followed by streptavidin-HRP (Southern Biotech) and developed with SIGMAFAST OPD tablets (Sigma-Aldrich). Absorbance values at 490 nm were determined and used to calculate endpoint titers.
For ELISpot, serial dilution of splenocyte suspensions of μMT chimeric mice were added to NP20-BSA-coated MultiScreen HA mixed cellulose ester plates (Millipore, Watford, UK) previously washed and blocked with IMDM (Sigma-Aldrich) freshly supplemented with 10% FCS, 2 mM GlutaMAX and 50 μM 2-mercaptoethanol. Upon overnight incubation, cells secreting anti-NP antibody (ASC) were visualized with HRP-conjugated anti-mouse IgM or IgG3 antibodies (Southern Biotech) followed by AEC staining Kit (Sigma-Aldrich). The numbers of ASCs were quantified using Immunospot S6 Analyzer (Cellular Technology Limited).

For ELISPOT, MultiScreen HA mixed cellulose ester plates (Millipore) were coated with NP₂₃-BSA (Biosearch Technologies) or NP₂-BSA (conjugated in-house) in PBS, washed, and blocked with complete medium before applying serially diluted cell suspensions and incubating overnight under 5% CO₂ at 37°C in IMDM, 10% FCS, 2 mM GlutaMAX, and 50 µM 2-mercaptoethanol. Cells secreting anti-NP antibody were visualized with horseradish peroxidase–conjugated anti–mouse IgG1 antibody (SouthernBiotech) followed by an AEC staining kit (Sigma). The numbers of ASCs were quantified using Immunospot S6 Analyzer (Cellular Technology Limited). ELISA was performed essentially as described previously (Newman et al., 2017 (link)).

For the quantitative ELISA assay of cytokines, sorted CD8⁺, CXCR5⁺, or CXCR5⁻CD8⁺ T cells were cultured with or without PMA and ionomycin for 48 h. The culture supernatants were collected for the quantitative assessment of IL-21 with Human IL-21 ELISA Ready-SET-Go!® (eBioscience), IFN-γ and IL-4 with BD OptEIA^TM Human ELISA Sets (BD Bioscience) according to the manufacturer's instructions. For the ELISA assay of immunoglobulins, co-culture supernatants from B cells and T cells were harvested, and the levels of IgG, IgM, and IgA were measured by Human IgG, IgM or IgA ELISA Ready-SET-Go!® (eBioscience). For the ELISPOT assay of cytokine-producing cells, sorted CD8⁺, CXCR5⁺ or CXCR5⁻CD8⁺ T cells were suspended in complete RPMI-1640 medium at a density of 1 × 10⁶/ml and stimulated with PMA plus ionomycin in pre-coated plates,100 μl/well for 16 h. The frequency of IL-21 or IFN-γ-producing cells was counted using an ImmunoSpot S6 Analyzer (Cellular Technology Ltd., USA) according to the manufacturer's instructions, and the results were shown as the mean of readings from triplicate wells.

Baloxavir susceptibilities were determined by using a focus reduction assay as previously described [1 (link)] in humanized MDCK cells (i.e., hCK cells), which express high levels of α2,6-sialoglycans and very low levels of α2,3-sialoglycans [15 (link)]. hCK cells were kindly provided by Dr. Yoshihiro Kawaoka (University of Wisconsin–Madison). hCK cells in 96-well plates were infected with 1000 focus-forming units (FFU)/well of viruses. Virus adsorption was carried out for 1 h at 37 °C and then an equal volume of 1.2% Avicel RC-581 (DuPont Nutrition USA, Wilmington, DE, USA) in culture medium containing serial dilutions (0.025–2500 nM) of baloxavir was added to each well in triplicate. The cells were incubated for 24 h at 34 °C and then fixed with formalin. After the formalin was removed, the cells were immunostained with a mouse monoclonal antibody against influenza A virus nucleoprotein (Merck KGaA, Darmstadt, Germany), followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (SeraCare Life Sciences, Milford, MA, USA). The infected cells were stained with TrueBlue Substrate (SeraCare Life Sciences) and then washed with distilled water. After cell drying, the focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology, Cleveland, OH, USA). The results are expressed as IC₅₀ values.

In the mouse breast cancer model, ICG-Lipo-PTX or saline was administered intraperitoneally, and PDT was performed only on the left-sided tumor according to the PDT protocol described above. After 14 days of treatment, mice were euthanized, and splenocytes were collected. ELISPOT assays were then used to detect interferon (IFN)-γ, interleukin (IL)-4, IL-2, and IL-10 in order to determine whether coculture with BALB-MC cells caused cytokine secretion to vary between the ICG-Lipo-PTX group and PDT-only group. Splenocytes (6.0 × 10⁵) and BALB-MC cells (6.0 × 10⁵) were added to the plates, and the plates were placed in an incubator containing 5% CO₂ for 24 h at 37°C. An Immunospot S6 Analyzer (Cellular Technology Limited, Cleveland, OH, USA) was used to automatically count the number of spots.

For analyzing the existence of peptide-specific memory T cells in unexposed healthy donors, IFN-γ ELISPOT assay was performed to assess the ex vivo response of PBMC to peptides, using ready-to-use human IFN-γ ELISPOT kit (Dakewe Biotech, Shenzhen, China). PBMCs were plated in triplicate with 2 × 10⁵ cells per well, with peptide added to ELIPSOT wells at 4 μM concentration. Plates were incubated overnight at 37 °C for 22 h. The following ELISPOT assay was performed according to the manufacturer’s instructions. The number of spot-forming units was determined on a C.T.L. ImmunoSpot S6 Analyzer and analyzed by ImmunoSpot v6.0 software (Cellular Technology Ltd., Cleveland, OH, USA).