Papaine

d‐glucose, calcium d‐gluconate, DMSO, ACh, ATP, mecamylamine, NaHCO₃, papaine, l‐cysteine, leupeptine, pyruvic acid, NaOH and tyrphostin AG490 (AG 490) were acquired from Sigma‐Aldrich (Taufkirchen, Germany). A‐85380, α‐bungarotoxin, α‐conotoxin PnIA, α‐conotoxin MII, chelerythrine chloride, dihydro‐β‐erythroidine (DHβE), epibatidine, T16Ainh‐A01, Ani 9, CaCCinh‐A01, Chromanol293B, ACV 1 (conotoxin Vc1.1), dantrolene, JTV519 and H‐89 were obtained from Tocris Bioscience (Abingdon, UK). The α‐conotoxin ImI was ordered from Alomone Labs (Jerusalem, Israel). KH₂PO₄, KCl and MgCl₂ were purchased from MERCK (Darmstadt, Germany). NaCl was ordered from Grüssing GmbH (Westoverledingen, Germany). HEPES and BSA were from Carl Roth (Karlsruhe, Germany). Thapsigargin and WP1066 were acquired from Cayman Chemicals (Ann Arbor, MI, USA) and nicotine from Glentham Life Sciences (Corsham, UK). EDTA and Tween 20 were obtained from VWR (Darmstadt, Germany). Sodium pyruvate was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA) and DNase1 from Invitrogen (Thermo Fisher Scientific).

Neurospheres were prepared as previously described^{37 (link)}. Briefly, E15.5 embryos were individually dissected in PBS and the neocortex was transferred in Dulbecco’s Modified Eagle Medium/F12 (DMEM) and dissociated by extensive enzymatic digestion with Papaine (Sigma-Aldrich). Cells were grown in medium containing DMEM/F12, 0.66% glucose, L-glutamine 1%, Pen/Strep 1%, 4 µg/mL of heparin, hormone mix with the addition of either 20 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic fibroblast growth factor (bFGF). In such conditions, cells spontaneously formed neurospheres. Neurospheres were then dissociated into single-cell cultures and plated on matrigel-coated coverslips (BD Bioscience).

Ribosomal RNA content per cell was quantified by calculating 18S, 5.8S and 28S rRNA RT-qPCR signals in six biological replicates relative to the DNA content. DNA concentrations in equal volumes of papain digestion buffer (100 mM phosphate buffer (NaH₂PO₄ (VWR, Amsterdam, the Netherlands) and Na₂HPO₄ * 2H₂O (VWR), pH 6.5), 5 mM l-cysteine*HCl (Sigma-Aldrich), 5 mM EDTA (Ethylenediaminetetraacetic acid)(VWR), 33.33 μg/μl papaine (Sigma-Aldrich)) were determined in samples from day 0, 7 and 14 of ATDC5 chondrogenic differentiation using the SYBR Green assay (Invitrogen). Prior to measurement, samples were diluted in TE (Tris-EDTA) buffer (10 mM Tris/HCl pH 8 and 1 mM EDTA; day 0; 1:100 dilution, day 7 and day 14; 1:1000 dilution). A serially diluted standard curve (0.016–4 μg/ml) of calf thymus genomic control DNA (Invitrogen) in TE buffer was included to quantify the DNA concentration in the samples. Standards were prepared to contain the same amount of papain digestion buffer as the samples. SYBR Green was diluted 10,000 times in TE buffer and 100 μl was added to 100 μl of the above prepared samples and standards. After 10 min incubation fluorescence was determined using a Spectramax M2E (Molecular Devices, Sunnyvale, CA, USA) microplate reader with an excitation of 488 nm and an emission of 522 nm and DNA concentration was calculated using the standard curve.