Phytohemagglutinin m pha m

Sorted ILC2s (100 cells/well) were expanded with human feeder PBMCs (100,000/well; 37 °C, 5% CO₂) for three to five weeks. Culture medium contained RPMI 1640 Glutamax medium (Life Technologies, Darmstadt, Germany), 1% Pen/Strep (Life Technologies, Darmstadt, Germany), 10% h.i. human AB serum (Sigma-Aldrich, Taufkirchen, Germany), and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Lonza, Wakersville, USA). The medium was supplemented with 100 U/ml rh-IL-2 (Life Technologies, Darmstadt, Germany), 25 ng/ml rh-IL-4 (Miltenyi Biotech, Bergisch Gladbach, Germany), 5 µg/ml phytohemagglutinin-M (PHA-M; Sigma-Aldrich, Taufkirchen, Germany).

γ-Hexalactone (CAS number 695–06-7), histopaque-1077^® and reagents for cell culture, including RPMI 1640 medium, fetal bovine serum (FBS), penicillin/streptomycin, phytohemagglutinin-M (PHA-M) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and obtained from standard commercial suppliers.

Splenocyte cells were re-suspended to a concentration of 2.5 × 10⁶/ml in complete RPMI and seeded at a density of 2.5 × 10⁵ per well in a 96 well ELISPOT plate that contained immobilized IFN-γ-specific monoclonal antibodies (mAb) from the IFN-γ ELISPOT kit (Mabtech, Ohio, USA). Wells that contained 5 µg/ml of purified phytohemagglutinin-M (PHA-M) (Sigma Aldrich, Calif., USA) served as positive control wells. The MMS and pIY vaccine strains or the IV, combined with Freund’s incomplete adjuvant were each added to the wells at a final concentration of 10⁴ PFU/ml for overnight stimulation. After 24 h, the cells were removed and the detection mAb which was biotinylated was added. Thereafter, a streptavidin-enzyme conjugate was added and after addition of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate, the number of spots were counted using the automated ELISPOT reader (Cellular Technology Limited, Ohio, USA) to determine the frequency of IFN-γ secreting cells.