Casy cell counter and analyser system model tt

Absolute cell counts from the mLN and TC of individual mice were determined using the CASY® Cell Counter and Analyser System Model TT (Roche Innovatis AG, Reutlingen, Germany). FACS staining was performed as previously described (Rodriogo et al. 2016 (link)). In brief, cells were fixed and permeabilised with intracellular fixation and permeabilisation buffer set (Thermo Fisher Scientific) according to the manufacturer’s instructions. Thereafter, cells were stained with combinations of fluorophores (FITC, PE, PE-Cy7, PerCp-Cy5.5, APC) conjugated with anti-mouse CD4, CD11b, Foxp3, F4/80, GR1, Ly6c, pStat3 (Y705), Rorγt (eBioscience) and IL-17A (Biolegend, San Diego, USA) monoclonal antibodies to determine distinct cell populations. Expression levels were determined using the FACS Canto flow cytometer (BD Bioscience) and analysed with FlowJo v10 software (FlowJo, LLC, USA).

Human embryonic kidney cells (HEK-293T, ATCC: CRL-11268) and immortalized hMSCs43 (link) were cultivated in Dulbecco’s modified Eagle’s medium (Invitrogen, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS; cat. no. F7524, lot no. 022M3395, Sigma-Aldrich, Munich, Germany) and 1% (v/v) penicillin/streptomycin solution (Sigma-Aldrich, Munich, Germany). HEK-293-derived FreeStyle 293F suspension cells (HEK-293F; Invitrogen) were grown in FreeStyle 293 expression medium (Invitrogen). All cell types were cultivated at 37 °C in a humidified atmosphere containing 5% CO₂. Cell concentration and viability were profiled with a CASY Cell Counter and Analyser System Model TT (Roche Diagnostics, Mannheim, Germany). For (co)-transfection, 5 × 10⁴ HEK-293T, HEK-293F or hMSCs were diluted in 0.4 ml of culture medium and seeded per well of a 24-well plate 12 h before (co)-transfection. The cells were then incubated for 6 h with 200 μl of a 1:2 PEI:DNA mixture (w/w) (polyethyleneimine; MW 40,000, Polysciences, Inc., Warrington, USA) containing 1 μg of total DNA (for co-transfections, an equal amount of plasmid DNA was used unless otherwise indicated). After (co)-transfection, the culture medium was replaced, and the engineered cells were used for a dedicated experiment, which was typically analysed for 48 h.