Hek293 cells

To generate receiver cells, HEK293 cells which express the puromycin-resistant mediating PAC transgene for later selection in the experimental setup were used. In a first step, the tTA or Gal4 binding motif 5′ of the CMV_min promoter (Response Element; RE) was transduced into HEK293 cells (German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany). Cells were seeded with a density of 25,000 cells/cm² in a 6-well culture dish (TPP, Switzerland), and 2 mL of viral supernatant containing 10 µM protamine sulfate (Merck, Darmstadt, Germany) was added to the cells. The medium was changed after 24 h and cultured for another 72 h. These HEK293 cells were passaged and reseeded with a density of 25,000 cells/cm² and transduced a second time with either the LaG17_synNotch_TetRVP64 (tTA RE), the antiCD19_synNotch_Gal4VP64, antiHer24D5-3_synNotch_Gal4VP64, or antiHer24D5-5_synNotch_Gal4VP64 (all Gal4 RE) viral supernatant (2 mL) containing 10 µM protamine sulfate. This double transduced, heterogeneous cell population was cultured for an additional 72 h and was then activated with their corresponding antigen (see below) and sorted by flow cytometry (see below). Of note, the maintenance culture was frequently analyzed for unspecific or spontaneously dsRed-Express expression.

Culturing of HEK293 cells and MEFs, double deficient for iRhom1 and iRhom2 (MEF_dKO): in a humidified incubator at 37 °C, 5% CO₂ in DMEM10%, which consists of DMEM high-glucose (Sigma-Aldrich) supplemented with 10% foetal calf serum (PanBiotech), 100 mg/l streptomycin (Sigma-Aldrich) and 60 mg/l penicillin (Sigma-Aldrich). Generating of stable cells was performed as described before [28 (link)]. MEFs were obtained from indicated mouse lines as described before [19 (link)]. BMDMs were freshly isolated from femur and tibiae of respective mouse lines as described previously [42 (link)]. HEK293 cells were purchased from German Collection of Microorganisms and Cell Cultures (GmbH DSMZ- No. ACC 305).