Mycoplasma detection kit

GH3 (CCL-82.1, January 2016) and AtT-20 cells (CCL-89, July 2014) were obtained from ATCC and grown in Ham’s F12-K Medium (Gibco, Life Technologies) supplemented with 15% Horse serum and 2.5% heat-inactivated FBS or in Ham’s F12-K Medium (Gibco) supplemented with 15 horse serum and 2.5% FBS, respectively. TtT/GF cells were obtained from RIKEN BRC (RCB1279, September 2019) and cultured in DMEM/HamF12 (Gibco) supplemented with 10% Horse serum and 2.5% FBS. Starvation medium resembles the growth medium but contains 0.5% Horse serum and 0.125% FBS (heat inactivated for GH3 cells). Routinely, all cell lines were tested for mycoplasma contamination by using Mycoplasma Detection Kit (minerva biolabs, Berlin, Germany). Identity of the cells was analyzed by marker gene expression analyses and immunofluorescent stainings against marker proteins as shown in Supplementary Fig. 3. Passage numbers between 15 and 30 of cell lines were used for the respective experiments.
Detailed information about SAG treatment, preparation of conditioned medium, medium transfer experiments, measurements of supernatant hormone/neuropeptide levels and BrdU incorporation analysis are given in Supplementary methods.

The conditional immortalized human podocytes, control podocytes and podocytes with reduced α-Gal activity were cultured as previously described¹³ . Briefly, immortalized podocytes were transduced with co-shRNA (control), and with shRNA 894 (α-galactosidase A knockdown) and cultured in RPMI media (Sigma-Aldrich, Taufkirchen, Germany) with insulin-transferrin-sodium selenite as supplement (ThermoFisher Scientific, Waltham, USA) containing 10% fetal bovine serum (Biochrom, Berlin, Germany). Cells proliferated at 33 °C until they reached a confluence of 60–70%. Afterwards cells were differentiated at 37 °C for 14 days before harvesting them for proteomic analysis. Cells were regularly tested for mycoplasma infection using mycoplasma detection kit from Minerva biolabs (Minerva Biolabs, Berlin, Germany).

Human iPSC line (Ctrl-2), derived from a healthy mid-age Caucasian female donor peripheral blood mononuclear cells (PBMCs), established and characterized earlier [32 (link),33 (link)], was used in this study. Cells were cultured on BD Matrigel™ matrix (BD Biosciences, Franklin Lakes, NJ, USA) with mTeSR™1 medium (Stem Cell Technologies, Vancouver, Canada), using Gentle Cell Dissociation Reagent for passages, according to the manufacturer’s instruction. Representative hiPSC colony morphology, pluripotency staining for POU5F1, NANOG, SSEA4, and TRA1-60 and karyotype analysis are presented in Figure S1. For mycoplasma screening, the Venor^®GeM-Advance (Minerva Biolabs) Mycoplasma Detection Kit was used according to the manufacturer’s protocol in every fifth passage during maintenance and before freezing. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. In the current study cultures from passage 16 and 17 were used for differentiation.

All cell lines were regularly tested for mycoplasma contamination via the Mycoplasma Detection Kit for conventional PCR (Venor^®GeM Classic, Minerva Biolabs, Berlin, Germany) using MB Taq Polymerase (5 Unit/µL, 50 Units). All tested cell lines were found to be mycoplasma-negative.

Colorectal cancer cell lines were grown under the standard conditions in DMEM/F12 medium (Thermo Fisher Scientific, Darmstadt, Germany) supplemented with 10% FCS in the presence of 100 U/mL penicillin and 100 µg/mL streptomycin (PAA Laboratories GmbH, Cölbe, Germany). HCT116 (ECACC#91091005), Colo 205 (ECACC#87061208) and HeLa (ECACC#93021013) cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC; https://www.phe-culturecollections.org.uk (accessed on 7 January 1997). HCT116 HygTK cell line was generated previously in our laboratory [15 (link)]. All cell lines were regularly checked for mycoplasm contamination using the Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany). Cell growth was determined by cell counting in a Neubauer hemocytometer.

¹⁴C-metformin (1,1-Dimethylbiguanide hydrochloride) and ³H-MPP (1-Methyl-4-phenylpyridinium iodide) were purchased from American Radiolabeled Chemicals, St. Louis, MO, USA. All non labelled chemicals were obtained from Sigma-Aldrich, Darmstadt, Germany.
With cDNA of hOCT2 (GeneBank: accession number: NM_003058.3) or mOct2 (NM_013667.2), stably overexpressing HEK293 cells were used for the experiments and empty vector-transfected HEK cells were used as control-HEK cells.
All HEK-293 cell lines (hOCT2-, mOct2 and the vector-HEK-cells) were routinely tested for mycoplasma using Mycoplasma Detection Kit (Venor^®GeM, Minerva biolabs, Berlin, Germany) for conventional PCR and used if free from mycoplasma.