Pyruvate

Cell culture material including media and supplements were from Biochrom (Berlin, Germany). Cells were cultured in a humidified atmosphere with 5% CO₂ at 37 °C. Monkey kidney (COS-7)and human embryonic kidney (HEK293) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% L-glutamine and 1% pyruvate (Biochrom).
Heterologous cells were transiently transfected using X-treme gene 9 transfection reagent (Roche, Mannheim, Germany) or FuGene6 (Promega, Mannheim, Germany) according to manufacturers´ protocols. For antibody staining, transfection of single constructs and co-localization studies with organelle markers, cells were grown on 13 mm glass coverslips and transfected with 1 μg of each construct. For endocytosis assay, HEK293 cells were transfected with 2 μg of each construct. Cells were incubated for 24 h after transfection before staining, fixation with 4% paraformaldehyde, or endocytosis assays with Alexa Fluor® 488 AcLDL.

A549 lung epithelial cells (ATCC^® CCL-185, American Type Culture Collection, Manassas, VA, USA) were cultivated in both laboratories according to standard cell culture procedures in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% foetal calf serum (FCS, PAN Biotech, Aidenbach, Germany), 1 mM pyruvate (Biochrom, Berlin, Germany) and 2 mM glutamine (Merck, Darmstadt, Germany) without antibiotics [41 (link)] and passaged twice a week. Mycoplasma contamination was controlled by qPCR, and cell culture A549 passages 5–30 were used for DHM QPI experiments.

PBMCs were isolated from 50 mL of peripheral blood obtained from healthy donors, by Ficoll-Paque Plus (Thermo Fisher Scientific) density gradient centrifugation followed by erythrocyte lysis using Buffer EL (Qiagen). After CD4 enrichment using magnetic cell separation with the autoMACS instrument (Miltenyi Biotec) using CD4 MicroBeads (Miltenyi Biotec), cells were left overnight at 4°C in basal cell culture medium [RPMI 1640 Medium with GlutaMAX Supplement-10 (Thermo Fisher Scientific), 10% FCS (Corning), 50 nM 2-mercaptoethanol (Thermo Fisher Scientific), 1 mM pyruvate (Biochrom), and 25 mM HEPES (Gibco)]. The following day, CD4⁺ memory T cells (CD4⁺ CD25⁺ CD127⁺ CD45RO⁺) and Tregs (CD4⁺ CD25⁺ CD127^–) were FACS-sorted with a BD FACSAria II SORP (Becton Dickinson). Sorted CD4⁺ memory T cells and Tregs were initially cultured in 96-well round-bottom plates (Greiner Bio-One) with the aforementioned cell culture media supplemented with 500 IU/mL rhIL-2 (R&D Systems) and 10 nM Rapamycin (STEMCELL Technologies) (day 0). The following day, cells were stimulated with anti-CD3/CD28 MACSiBead particles (Treg Activation/Expansion Kit, Miltenyi Biotec) according to manufacturer’s guidelines. Cells were stimulated at the same time points as the 1st and 2nd generation products, and collected for downstream DNA methylation analysis.