β mercaptoethanol

Splenocytes from immunized and control mice were isolated after the second immunization. Before staining, splenocytes were grown in RPMI 1640 (EuroClone) containing 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies), 100 U/mL penicillin (EuroClone), 100 U/mL streptomycin (EuroClone), 100 μg/mL glutamine (EuroClone), 0.1 mM non-essential amino acids (EuroClone), 1 mM sodium pyruvate (EuroClone), 50 μM β-mercaptoethanol (EuroClone) and 10 U/mL IL-2 (Miltenyi Biotec). Cells were stimulated for 4 h with 0.25 μM ionomycin and 10 ng/mL phorbol myristate acetate (PMA) and 2 hours with 10 μg/ml brefeldin A. A total of 1.5 × 10⁶ cells were stained in PBS 1X/0.5% bovine serum albumin/2 mM EDTA for 10 min at 4°C with the following antibodies (Miltenyi Biotec): anti-CD3 Vioblue; anti-CD4-APC, anti-CD8-PE-Vio770, anti-CD62L-FITC and anti-CD49d-PE for T cell detection. The cells were then fixed and permealized using the Miltenyi Biotec Inside Stain Kit and intracellularly stained according to the manufacturer’s instructions with IFN-γ-PE or APC (Miltenyi Biotec). The CD8 + CD3+; CD3 + CD4+ cells were gated and then analyzed by flow cytometry for IFN-γ production and for the evaluation of CD62L and CD49d expression. Flow cytometry acquisition was performed on a MACSQuant instrument, and the data were analyzed with the MACSQuantify Software (Miltenyi Biotec).

The LY27805 cell line was kindly provided by Bruno Amati’s group [7 (link)] at the European Institute of Oncology–Italian Foundation for Cancer Research (FIRC) Institute of Molecular Oncology (IEO–IFOM, Milan, Italy) campus, expanded and stored according to their instructions. Specifically, LY27805 cells were plated at 2 × 10⁵ cells/mL in B cell medium: 50:50 mixture of DMEM and IMDM (Euroclone, Pero (MI), Italy), 10% FBS (Euroclone, Pero (MI), Italy), 2 mM L-glutamine (Euroclone, Pero (MI), Italy), 1% penicillin/streptomycin (Euroclone, Pero (MI), Italy), 50 μM β-mercaptoethanol (Euroclone, Pero (MI), Italy), and non-essential amino acid (NEEA) (Euroclone, Pero (MI), Italy). Phoenix-Ampho cells were cultured with DMEM (Euclone, Pero (MI), Italy), 10%FBS (Euclone, Pero (MI), Italy), 2 mM L-glutamine (Euclone, Pero (MI), Italy), 1% penicillin/streptomycin (Euclone, Pero (MI), Italy).
Cells were tested and authenticated by the StemElite ID System (Promega, Madison, WI, USA). Cells were tested every six months for Mycoplasma by means of the ATCC Universal Mycoplasma Detection Kit 30-1012, cultured for no more than two weeks, and used for no longer than 15 passages.