Fetal bovine serum gold

BM-derived DC (BMDC) from C57/Bl6 mice were generated from precursor cells as described before (Lutz et al, 1999 (link)). In brief, 2 × 106 BMDC per 10-cm dish (BD Falcon) were cultured for 8 d in R10 medium consisting of RPMI1640 (Lonza), 1% penicillin/streptomycin/L-glutamine (Sigma-Aldrich), 2-ME (50 µM; Sigma-Aldrich), and 10% heat-inactivated FBS (Fetal Bovine Serum Gold; GE Healthcare) and additionally supplemented with GM-CSF supernatant (1:10) from a cell line stably transfected with the murine GM-CSF (Zal et al, 1994 (link)). At days 3 and 6, 10 ml of fresh R10 supplemented with GM-CSF supernatant (1:10) was added, by removing 50% of the old cell culture supernatant at day 6 before. Maturation of BMDC was induced at day 8 by the addition of 0.1 ng/ml LPS (Sigma-Aldrich) for 20 h. At day 9, cells were used for further experiments.

Locust primary brain cell cultures were established from 4^th stage juvenile locusts as previously described (Ostrowski et al., 2011 (link); Miljus et al., 2014 (link)). Complete growth medium consisted of L15 (Leibovitz’s L-15 Medium, #11415049, Thermo Fisher Scientific, Germany), 5% FBSG (Fetal Bovine Serum Gold, PAA Laboratories GmbH, Austria), 1x Penicillin-Streptomycin (Penicillin-Streptomycin, 10,000 units penicillin and 10 mg streptomycin/ml, #P4333, Sigma-Aldrich^®, Germany) and 1% Amphotericin B (Gibco^TM Amphotericin B, 250 μg/ml, #15290018, Thermo Fisher Scientific, Germany). Dissected brains were pooled (see below), enzymatically digested with 2 mg/ml Collagenase/Dispase solution for 30–45 min at 27°C and mechanically dissociated by trituration with a 100 μl tip of an Eppendorf pipette. The primary brain cells were cultured on ConcanavalinA-coated (Sigma-Aldrich^®, Germany) round glass cover slips (Ø 10mm, Corning, Inc., Sigma-Aldrich^®, Germany) in 4-well NUNC plates (#176740, NuncTM Delta Surface, Thermo Fisher Scientific, Germany) filled with 500 μl of complete growth medium at 27°C in a humidified atmosphere. The medium was changed every 2 days. Based on previous studies (Gocht et al., 2009 (link)), locust brain cultures are estimated to contain approximately 3% glia and 97% neurons after 7 days in vitro under normoxic conditions.

Human embryonic kidney (HEK) cell line 293 T was maintained in Dulbecco’s modified Eagle’s medium (Invitrogen Life Technologies, Carlsbad, CA, USA). CRC cell line HT29 was cultivated in McCoy’s + GlutaMAX medium and CaCo2 cell line was maintained in MEM + GlutaMAX Medium. The medium contained 10% (v/v) Fetal Bovine Serum Gold (PAA Laboratories GmbH, Pasching, Austria) and 1% (v/v) Penicillin/Streptomycin (Invitrogen Life Technologies). All cell lines were cultured in a 5% CO₂-humidified incubator (Nalge Nunc International, Rochester, NY, USA) at 37°C.
The Pre-miR miRNA Precursor of miR-518a-5p (Ambion Life Technologies, Carlsbad, CA, USA; Cat. No. 17100 ID: PM12865) and the negative control (Ambion Life Technologies, Cat. Nr. 17110) were purchased from Ambion Life Technologies.

HeLa cells (obtained from DSMZ) were grown in Minimum Essential Medium with Earle’s salt (DMEM, Life Technologies) supplemented with 10% Fetal Bovine Serum Gold (PAA Laboratories), 10 mM Hepes (Sigma) and 1% NEAA (Life Technologies) under standard conditions. Primary skin fibroblasts from SCA2 patients and age- and sex- matched healthy control individuals (CTL) described previously [54 (link)] were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 4.5 g/l glucose, 2 mM L-glutamine, 100 U/ml penicillin G (all Life Technologies), 100 μg/ml streptomycin (Life Technologies) and 10% Fetal Bovine Serum Gold (PAA Laboratories).
HeLa cells were transfected with one or two of the plasmids stated below. Therefore, 200,000 cells per 6-well or 1.5 million cells per 10-cm dish were seeded 16 h prior to transfection. Transfection was conducted with Effectene Transfection Reagent Kit (Qiagen) using 1 μg (for 6-well) or 5 μg (for 10-cm dish) plasmid DNA according to the manufacturer’s instructions. The transfected cells were incubated for 48 h.

Human embryonic kidney cells (HEK293) and normal human dermal fibroblasts (NHDFs) were obtained from the American Type Culture Collection (Rockville, MD) and maintained in high-glucose DMEM (PAA Laboratories, Pasching, Austria) supplemented with 10% (vol/vol) fetal bovine serum Gold (PAA Laboratories) at 37°C in a 5% (vol/vol) CO2 atmosphere.

Human embryonic kidney cells (HEK293, CRL-1573) were purchased from the American Type Culture Collection (Rockville, MD, USA) and cultivated in high-glucose DMEM (Dulbecco’s Modified Eagle Medium) (PAA Laboratories, Pasching, Austria) supplemented with 10% (v/v) fetal bovine serum Gold (PAA Laboratories) at 37 °C in a 5% (v/v) CO₂ atmosphere.