Ctl immunospot profession software

Antigen-specific PBMCs secreting IL-4 or IFN-γ were detected using a Human IFN-γ/IL-4 Double-Color ELISPOT (ImmunoSpot, Shaker Heights, Cleveland, OH), per manufacturer’s protocol. Briefly, cryopreserved PBMC cells were thawed, and 1-3 x 10⁵ cells were stimulated for 48 hours with 11 SARS-CoV-2 Spike peptide pools (17- or 18-mers with 11 amino acid overlap) (Genscript, Piscataway, NJ) at a concentration of 1μg/mL per peptide. DMSO and Concanavalin A (ThermoFisher, Waltham, MA), were used as negative and positive controls, respectively, as previously described (12 (link)). Spots were counted on an Immunospot Analyzer with CTL Immunospot Profession Software (Cellular Technology Ltd., Shaker Heights, Cleveland, OH). Spot forming cells (SFC) in peptide stimulated wells were computed following subtraction of SFCs detected in DMSO stimulated control wells and were considered positive if the number of SFC was > 1 spot per 1 x 10⁵ plated cells.

PBMC’s were stimulated in duplicate at 2e5 and 1e5 cells per well with 10 individual peptide pools (1μg/mL each peptide) spanning the full amino acid sequences of HA, NP, and M2e. HA and NP peptides were 15-mers overlapping by 11 amino acids (BEI Resources), M2e peptides were 10-mers overlapping by 9 (Mimotopes). Each pool contained 14–27 individual peptides and sequentially spanned the entire protein. Concanavalin A (Sigma) was used as a positive stimulation control (5μg/mL). DMSO served as a negative solvent control. Antigen-specific T cells secreting IFN-γ were detecting using paired anti-macaque IFN-γ monoclonal antibodies (U-cytech-BV) as previously described. Spot forming cells (SFC) were enumerated using an Immunospot Analyzer with CTL Immunospot Profession Software (Cellular Technology Ltd.). Results are expressed as the mean number of SFC in replicate wells containing antigenic peptide, subtracting the number of spots from DMSO control wells from the same animal.

Antigen-specific T-cells secreting IFN-γ in the PBMCs were detected using a Human IFN-γ Single-Color ELISPOT or Human IL-4/IFN-γ Double-Color ELISPOT (ImmunoSpot, Shaker Heights, Cleveland, OH), per the manufacturer’s protocol. Briefly, cryopreserved PBMC cells were thawed, and 1–3 x 10⁵ cells were stimulated for 24–48 hours with 11 SARS-CoV-2 Spike peptide pools (17- or 18-mers with 11 amino acid overlap) (Genscript, Piscataway, NJ) at a concentration of 1μg/mL per peptide. DMSO was used as a negative control and Concanavalin A (ThermoFisher, Waltham, MA) or Phorbol 12-myristate 13-acetate (PMA) and Ionomycin (Sigma-Aldrich, St. Louis, MO) were used as positive controls, as previously described (11). Spots were counted on an Immunospot Analyzer with CTL Immunospot Profession Software (Cellular Technology Ltd., Shaker Heights, Cleveland, OH). Spot forming cells (SFC) were computed following DMSO subtraction and were considered positive if the number of SFC was > 10 SFC per 1 x 10⁶ cells.