Hygromycin b

Rat Park2 single guide RNA (sgRNA), rat Pink1 sgRNA, and Cas9 were designed by and purchased from Toolgen (Republic of Korea). The target sequence for Park2 was 5′-ATCACTCGCAGCTGGTCAGCTGG-3′ and the target sequence for Pink1 was 5′-CCTGACACCGGGCCCGGCTTGGG-3′. HCN cells were transfected with Park2 sgRNA or Pink1 sgRNA and Cas9 for 24 h and selected by hygromycin B (InvivoGen, San Diego, CA, United States).

Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fisher Scientific) containing 25 mM D-glucose, 1 mM sodium pyruvate supplemented with 10% heat-inactivated fetal calf serum (FCS), (Biochrom, Berlin, Germany), 1% Penicillin/Streptomycin (Thermo Fisher Scientific), and 140 μg/mL Hygromycin B (Invivogen, Toulouse, France) in a humidified atmosphere of 5% CO₂ at 37°C.
For simultaneous determination of TLR4 stimulation and viability, a novel monoclonal dual reporter cell line was established. Therefore, the HeLa TLR4 cell line (Novusbio, Wiesbaden, Germany), expressing Renilla luciferase under the control of an IL-8 promotor reporting TLR4 activity, was stably transfected with a plasmid, constitutively expressing Firefly luciferase, and, thereby, measuring viability (pCMB-firefly-luc-hygro, kindly provided by Ernesto Bockamp, University Medical Center of the Johannes Gutenberg University). Lipofectamine 3000 (Thermo Fisher Scientific) was used as the transfecting reagent according to the manufacturer's protocol.

Stable HEK293 Flp-In/T-Rex cells (Invitrogen) for the inducible production of mIL-6-RFP-Fc were generated with the Flp-In system using 250 μg/ml hygromycin B (Invivogen) and 15 μg/ml blasticidin (Invivogen). Protein expression was induced in serum-free medium with 400 ng/ml doxycycline (Sigma-Aldrich) for 72 h. Harvested conditioned media were cleared through centrifugation, passed through a 0.20 μm sterile-filter and afterwards loaded onto an ÄKTA Purifier 10 system (GE Healthcare, Chalfont St. Giles, UK) for affinity purification on a 1 ml Protein A Sepharose Column (#89924, Thermo Fischer, Waltham, MA). The column was washed with 20 mM sodium phosphate buffer (pH 7). mIL-6-RFP-Fc was eluted in fractions of 1 ml using 12.5 mM citric acid (pH 2.7) and neutralized immediately by adding 50 μl of 2 M Tris/HCl (pH 8). Protein containing fractions were quantified using a BCA colorimetric assay (#500-0006, Bio-Rad, Munich, Germany) following manufacturer’s instruction. Protein containing fractions were pooled and dialyzed against PBS.

Mouse MEFs were reprogrammed into iPSCs as described.^{66 (link)} The CAG Pr-rtTA-3 × Stop-TRE-Runx1-p2a-Hoxa9-pA-PGK Pr-HygroR cassette was inserted into the Rosa26 locus of mouse ESC/iPSC. The positive clones (iR9-ESC/iPSC) selected by Hygromycin B (150 μg/mL, Invivogen) were further cultured in ES medium supplemented with Dox (1 μg/mL). The induced expression of Runx1 and Hoxa9 was confirmed by qPCR. For the generation of OT1-iR9-iPSC, a CAG Pr-OT1 TCRα-p2a-TCRβ-IRES-GFP-PGK Pr-PuroR cassette was inserted into the Hipp11 locus of iR9-iPSC. The OT1 sequence was cloned from murine TCR OT1-2A.pMIG II (Addgene). The OT1-iR9-iPSC-positive clones were further selected by Puromycin (1 μg/mL, Invivogen) and the expression of OT1-TCR was measured by intra-cellular staining.