Dmem low glucose medium

Rat sarcoma XC cells (ATCC Cat# CCL-165, RRID:CVCL_1891) were cultured in DMEM Low-Glucose medium (Sigma-Aldrich, St. Louis, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco Lab., New York, USA). Media were supplemented with 100 IU/ml penicillin and 10 µg/ml streptomycin (Polfa, Tarchomin, Poland) and cells were cultured in a humidified atmosphere with 5% CO₂ at 37 °C. For transfection experiments, cells were seeded into the wells of a 24-well plate at a density of 8 × 104 cell/well and cultivated for 24 h in media with 10% FBS without antibiotics (to reach 70–80% confluence).
PTAI-6–8/DOPE and PTAI-10–14/DOPE/DC-cholesterol mixtures were diluted in serum‐free and antibiotic-free cell culture medium or 5% glucose and mixed with pEGFP‐C1 plasmid diluted in cell culture medium or 5% glucose, respectively, to final concentration of 0.2 μg/μl. Next, they were incubated for 30 min at room temperature to obtain lipoplexes and diluted in serum‐free DMEM F12 HAM medium (1:4). 2.5 μg of PTAI- 6–8/DOPE and 4 μg of PTAI-10–14/DOPE were used for transfection of cells per well of 24-well plate. Lipids and pDNA were mixed at calculated ± charge ratio of 1:2.0 for PTAI-10–14 and 1:2.3 for PTAI-6–8-based lipoplexes.

Gold nanospheres 5 nm (product code # 752568), 50 nm (# 753645) and 100 nm (# 753688) as well as Aβ42 peptide were purchased from Sigma Aldrich, USA. The AuNSs were stored at 4 °C and used as supplied. Ni–NTA agarose was obtained from Qiagen. IPTG and PMSF were purchased from Sigma Aldrich, USA. Urea was purchased from Calbiochem, India. All other chemicals were of analytical grade and were procured from Merck, India. Horse Serum was bought from Himedia Laboratories. Fetal Bovine Serum (FBS), trypsin–EDTA, penicillin and sterptomycin were obtained from Gibco (Thermo Fisher Scientific). Thiazolyl Blue, tetrazolium bromide (MTT), DMEM low glucose medium and Nerve Growth Factor-7S were purchased from Sigma Aldrich.

As the source for TM proteins, the immortalised human TM cell line HTM5 and the glaucomatous TM cell line GTM3 were used. Both cell lines were a kind gift of Professor Abbot F. Clark and colleagues [North Texas Eye Research Institute (NTERI), Fort Worth, TX, USA]. The cell lines are derived from human TM as described elsewhere.40, 67 Cells were grown in DMEM low glucose medium (Sigma‐Aldrich, St. Louis, MO, USA), supplemented with 10% foetal calf serum (Gibco, Life Technologies, Carlsbad, USA), 1% penicillin/streptomycin solution (Sigma‐Aldrich, St. Louis, MO, USA) and 1% 200 mm L‐alanyl‐L‐glutamine (Biochrom, Berlin, Germany). The TM cells were cultured in an incubator at 37°C and 5% CO₂. Media were changed every 2–3 days. On confluence, cells were collected using cell scrapers. Cell pellets were snap‐frozen in liquid nitrogen and stored at −20°C until further use.

We used primary BTICs that were isolated from freshly resected human gliomas, as described (10). Specimen sampling and BTIC culture were approved by the Ethics Committee of the University of Regensburg (No° 09/101) and all patients gave written informed consent. BTICs were kept in DMEM low glucose medium (DMEM with 1 g/L of glucose; Sigma-Aldrich, St. Louis, MO, USA, #D6046) containing Epidermal Growth Factor (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-097-751) and Fibroblast Growth Factor (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-093-842) supplemented with 50 U (v/v) Penicillin, 0.05% (v/v) Streptomycin (#P4333), 2 mM (v/v) L-Glutamine (#G7513), 1% (v/v) MEM Vitamin Solution (#M6895), and 1% (v/v) non-essential amino acids (#M7145) (all Sigma-Aldrich, St. Louis, MO, USA). For differentiation, growth factors were withdrawn, and cells were exposed to 10% fetal calf serum (FCS) for at least two weeks. Cells were incubated at 37 °C, 5% CO₂, 95% humidity in a standard tissue culture incubator.

TK6 cells were obtained from the European Collection of Cell Culture (ECACC, Cat. 95111735). Cells were cultivated in RPMI 1640 culture medium (Sigma) supplemented with 10 % heat-inactivated fetal bovine serum (FBS, 20 min, 55 °C), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere (5 % CO₂ and 37 °C).
Chinese hamster lung fibroblasts (V79-4) obtained from the European Collection of Cell Culture (ECACC, Cat.93010723) were cultured in flasks in DMEM low glucose medium (Sigma), with activated 10 % FBS, 100 U/ml penicillin and 100 μg/ml streptomycin and 200 mM L-glutamine in a humidified atmosphere (5 % CO₂ and 37 °C). Both cell lines were tested for Mycoplasma with the MycoAlert™ PLUS Mycoplasma Detection Kit, before use. This kit detects the activity of mycoplasmal enzymes (detects all the main mycoplasma contaminants). Tests were performed after the first passage and no mycoplasma contamination was detected.

Fibroblasts were detached from culture dishes using TrypLE Express (Invitrogen, Waltham, MA, USA), centrifuged and suspended once more in the medium and quantified by One Scepter (Millipore, Billerica, MA, USA). Cells were again suspended in DMEM low glucose medium (Sigma-Aldrich, St. Louis, MO, USA) with 1% of human serum obtained from each subject, reaching a cell concentration of 500,000 per 1 mL. A solution was made by mixing the resuspended cells with plasma and agarose at 3% to create fibrin agarose/gels⁵ . 300 microliters of the solution were placed in the Transwell system (Corning, Midland, NC, USA) following the air-liquid technique^{3 (link)}. Culture medium was placed covering the entire substitute. Each system was incubated at 37°C in a 5% CO₂ atmosphere and with a relative humidity of 95% for 10 days (Figure 1a, b).
Figure 1Figure 2

Rat glomerular MCs were obtained from China Center for Type Culture Collection (no. HBYZ-1; CCTCC, WuHan, CN). Cells were cultured in DMEM low glucose medium (Hyclone, Logan, UT) with 10 % heat-inactivated FBS (Hyclone, Logan, UT) and 1 % antibiotic solution (Sigma-Aldrich, St. Louis, MO). Cells from passages three to five were used for all the experiments. Monolayer rat glomerular MCs were rendered quiescent in DMEM low glucose medium containing 0 % fetal bovine serum for 24 h. After treated with or without 30 mM D-glucose, mannitol or L-glucose (Sigma-Aldrich, St. Louis, MO), the cells were harvested for the extraction of protein and RNA. The ERK specific inhibitor PD98059 (Sigma-Aldrich, St. Louis, MO) was added to the cells 30 min before treated with 30 mM D-glucose, mannitol or L-glucose. All treatments were performed in DMEM low glucose media containing 0 % FBS. Each experiment was performed in triplicate.