Histopaque 1077 density gradient cell separation medium

PBMCs were isolated from ethylene diamine tetraaceticacid (EDTA) whole blood using LeucosepTM tubes (Greiner Bio-one International GmbH) and Histopaque®-1077 density gradient cell separation medium (Sigma-Aldrich) according to the manufacturers’ instructions. The ImmunoSpot® Human IFN-γ Single-Color Enzymatic ELISpot Assay Kit was utilized according to the manufacturer’s protocol (Cellular Technology Limited). PBMCs were plated at a concentration of 300,000 cells per well and were stimulated with six contiguous peptide pools spanning the length of the G protein sequence at a concentration of 2 µg/mL per peptide. One peptide (sequence AFNTVIALLGSIVII) was excluded due to false positive results. Analysis was performed using the CTL ImmunoSpot® Analyzer and ImmunoSpot® Software (Cellular Technology Limited). Spot forming units (SFU) per 1.0 × 10⁶ PBMCs were summed across the 6 peptide pools for each animal.

PBMCs were isolated from ethylene diamine tetraacetic acid (EDTA) whole blood using LeucosepTM tubes (Greiner Bio-one International GmbH, Frickenhausen, Germany) and Histopaque^®-1077 density gradient cell separation medium (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturers’ instructions. IFN-γ ELISpot assay of PBMCs was performed using the ImmunoSpot^® Human IFN-γ Single-Color Enzymatic ELISpot Assay Kit according to the manufacturer’s protocol (Cellular Technology Limited, Shaker Heights, OH, USA). PBMCs were plated at a concentration of 300,000 cells per well and were stimulated with two contiguous peptide pools spanning the length of the SARS-CoV-2 S protein sequence at a concentration of 2 µg/mL per peptide (Mimotopes, Mulgrave, Australia). Imaging was performed using the CTL ImmunoSpot^® Software (Cellular Technology Limited, Shaker Heights, OH, USA). Spot forming units (SFU) were hand counted and calculated per 10 [6 (link)] PBMCs as summed across the peptide pools for each animal.

PBMCs were isolated from ethylene diamine tetraaceticacid (EDTA) whole blood using LeucosepTM tubes (Greiner Bio-one International GmbH) and Histopaque®-1077 density gradient cell separation medium (Sigma-Aldrich) according to the manufacturers’ instructions. IFN-γ ELISpot assay of PBMCs was performed using the ImmunoSpot® Human IFN-γ Single-Color Enzymatic ELISpot Assay Kit according to the manufacturer’s protocol (Cellular Technology Limited). PBMCs were plated at a concentration of 300,000 cells per well and were stimulated with two contiguous peptide pools spanning the length of the SARS-CoV-2 S protein sequence at a concentration of 2 μg/mL per peptide (Mimotopes). Imaging was performed using the CTL ImmunoSpot® Software (Cellular Technology Limited). Spot forming units (SFU) were hand counted and calculated per 10⁶ PBMCs as summed across the peptide pools for each animal.