Staphylococcal enterotoxin a sea

Lymphocyte-enriched mononuclear cells were isolated by gradient separation from heparinized blood in Nycoprep 1.077A (Axis-Shield PoC AS, Dundee, UK) tubes centrifuged at 800g for 30 min at 20°C. After washing twice in PBS with 0.1% human albumin (Grifols Therapeutics, Inc., Research Triangle Park, NC, USA), cells were incubated with 1 μM fluorescent cell staining dye carboxyfluorescein diacetate succinimidyl ester (CFSE) using the CellTrace CFSE Cell Proliferation Kit (Invitrogen, Carlsbad, CA, USA). Duplicates of 4x10⁵ cells were added to each well of a round bottom 96-well plate containing either 0, 1, or 100 pg/mL of staphylococcal enterotoxin A (SEA; Sigma, St. Louis, MO, USA) and cultured for 7 days. Cells were harvested and labeled with an AlexaFluor 647-conjugated monoclonal anti-canine CD18 antibody CA1.4E9 (AbDSerotec, Raleigh, NC, USA) and analyzed by flow cytometry for CD18 expression (FL4 channel) and CFSE staining (FL1 channel, BD FACSCalibur, Becton-Dickinson, San Jose, CA, USA).