Complete rpmi 1640 medium

T-ALL cell lines (CUTLL1, DND41, JURKAT E6, RPMI-8402, TALL1, KOPTK1) were cultured in complete RPMI-1640 medium (EuroClone, Pero, Italy) supplemented with 10% FBS at 37 °C under 5% CO₂. UP-ALL13 cells [44 (link)] were cultured in complete RPMI-1640 medium supplemented with 20% FBS. HEK-293T were cultured in complete DMEM medium (EuroClone), supplemented with 10% FBS. Primary leukemia cells (AD#1) were obtained from the peripheral blood (PB) of a male patient at diagnosis. Informed consent and approval by the Azienda Ospedaliera di Padova Review Board were obtained according to general guidelines, conforming with the Declaration of Helsinki.

Lewis rats were immunized with 200 μg of R97-116 peptide (CASLO), the immunogenic region of rat AChR α-subunit, in CFA. Draining lymph nodes were aseptically removed 10 days post immunization, and LNCs suspensions were stimulated with R97-116 (10 μg/ml) in complete RPMI-1640 medium (Euroclone), containing 1% Na-pyruvate, 1% non-essential amino acids, 1% L-glutamine, 1% penicillin-streptomycin, 50 μM 2-mercaptoethanol, 2% normal rat serum (19 (link)). Antigen specific Teff were maintained by restimulation with R97-116 peptide every 15 days, and expanded with IL2 (10 U/ml) every 3 days.

For the in vitro experiments, whole blood samples from healthy donors were collected in EDTA-tubes by the Department of Transfusion Medicine (University Hospital of Udine) after a written informed consent was signed.
PBMCs were separated by centrifugation at 700 g for 20 minutes on a Ficoll Hypaque density gradient (Millipore) and resuspended at 1 x 106 cells/ml in RPMI 1640 complete medium supplemented with 10% uFBS, 1% glutamine, 1% Na pyruvate, 1% non-essential aminoacid, 1% penicillin/streptomycin, 1% Hepes and 50μM β-mercapthoethanol (all from Euroclone).
CD4+ T cells were purified from PBMCs by negative selection using a human CD4+ T cell enrichment kit (StemCell Technologies), according to the manufacturer’s instructions. Purity of monocytes was over 95% as proved by staining with anti-CD14 mAb (eBiosciences) and flow cytometry analysis (FACScalibur cytometer, BD, Biosciences).
Treg cells were isolated from PBMC with human CD4+CD25+CD127dim/- Regulatory T cell isolation kit II (Miltenyi Biotec). Purification efficiency was over 95%.
To remove monocytes, PBMC were stained with an anti-CD14 fluorescent antibody (clone 61D3, PE conjugated, eBiosciences) and CD14 negative cells were sorted by FACSAria II cell sorter (BD Biosciences). As a control, unfractioned PBMCs were sorted as well.