Cysteine hcl

After chondrogenic differentiation, the GAG and DNA content of the hydrogels was determined to evaluate cartilage-like matrix formation. After 1 and 28 days of culture, the GelMA discs were cut in half. One half of each disc was weighed, freeze-dried and weighed again prior to digestion using 200 µL papain digestion buffer (0.2 M NaH₂PO₄ + 0.01 M EDTA, pH = 6.0, supplemented with 250 µg/mL papain (P3125, Sigma-Aldrich) and 1.57 mg/mL Cysteine HCL (C9768, Sigma-Aldrich) overnight at 60 °C.
For determination of GAG concentration, papain-digested samples were diluted in PBS-EDTA and 46 µM 1,9-Dimethyl-Methylene Blue (DMMB, Sigma-Aldrich) solution was added to the samples. Absorption was measured at 525 and 595 nm on a spectrophotometer [66 (link)] and the ratio was used to calculate the concentration with a chondroitin sulphate C (Sigma-Aldrich) standard curve.
DNA concentration was measured in the papain digests with Picogreen (Quant-iT, Thermo Fisher Scientific) according to the manufacturers’ instructions. In short, samples were diluted 1:20 in TE-buffer (0.5 M EDTA in 1 M Tris (1:5), pH 8) and 100 µL sample was mixed with 100 µL Picogreen reagent, incubated 5 min in the dark and fluorescence was measured with an excitation of 485 nm and emission of 520 nm. Known concentrations of λDNA were used as a reference.

Primary cultured neurons were prepared from pregnant SD rats at the gestational age of 16–18 days. The procedures were all in accordance with ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357). Pregnant SD rats were anaesthetized by intraperitoneal injection of 10% chloral hydrate, and fetal rats were obtained by the cesarean. The fetal cerebral cortices were dissected with 0.5 mM EDTA and 0.5 mM cysteine-HCl (Sigma) in Earle's balanced salt solution, and then the cortices were gently triturated. The triturated cortices were digested at 37°C for 15–25 min with 1 mg/ml papain (0.5–2 units/mg, Sigma) before dissociated cells were suspended in the plating medium (Minimum Essential Medium containing 10% fetal bovine serum, 100 IU/ml penicillin and 100 IU/ml streptomycin). The number of dissociated cells was counted. 2–5 ml of cell suspension was planted on the coverslips (Sigma) in 24-well plates at a density of 1.5–3.0×10⁵ cells/ml. Cells were incubated at 37°C in a 95% O₂, 5% CO₂ humidified incubator for 4–6 h. Then the plating medium was replaced with the culture medium (Neurobasal A (Gibco BRL) medium containing 2% B27 and 0.5 mM L-glutamine (Gibco BRL)). The culture medium was replaced every 3–4 days.

The applied protective media consisted of 0.1 M phosphate buffer (PB) containing the protective agents sucrose (VWR International AG, Switzerland), inulin (RPN Foodtechnology AG, Switzerland), and glycerol (VWR International AG). Prior to preparation, components of PB and the used protectants were stored in an anaerobic chamber (10% CO₂, 5% H₂, 85% N₂) (Coy Laboratories, USA) overnight to remove residual oxygen. To prepare PB, sodium dihydrogen phosphate (6.0 g liter⁻¹) and sodium hydrogen phosphate (7.1 g liter⁻¹) (both from Sigma-Aldrich Chemie GmbH, Switzerland) were dissolved in oxygen-free distilled water. The pH was adjusted to 6.8 after the addition of the reducing agents cysteine-HCl and riboflavin (Sigma-Aldrich Chemie GmbH) at final concentrations of 1 g liter⁻¹ and 0.3 g liter⁻¹, respectively, to counter potential oxygen exposure during processing and storage (28 (link), 70 (link)). All three protectants (glycerol [15% {vol/wt}] and sucrose and inulin [both at 5% {wt/vol}]) (GSI) were dissolved in PB for cryopreservation, whereas a protective medium containing only sucrose and inulin (both 5% [wt/vol]) (SI) was used in the lyophilization trials. Protective media were filter sterilized, covered in aluminum foil for protection from light, and stored in an anaerobic chamber before use.

The 20 bifidobacterial strains used in this study (Table 1) had previously been isolated from infant faeces [49 (link), 50 (link)] except in two cases where the strains originate from adult faeces, and deposited in the APC Culture Collection (APC Microbiome Institute, Ireland). Bifidobacteria were routinely grown on de Man-Rogosa-Sharpe (MRS) medium (Difco Laboratories, Detroit, MI) supplemented with 0.05% w/v cysteine-HCl (Sigma, St. Louis, MO) (MRSC) and incubated at 37 °C under anaerobic conditions (10% H₂, 10% CO₂, and 80% N₂) in a chamber Mac 500 (Don Whitley Scientific, West Yorkshire, UK). For solid medium, 2% (w/v) agar (Oxoid, Basingstoke, UK) was added. Prior to each DNA extraction, bacteria were sub-cultured twice and overnight cultures were used.

B. castoris isolates representative of the 12 strains identified through genomic analysis, as well as B. longum NCIMB 8809 (negative control) were grown in unsupplemented mMRS and mMRS supplemented with either 0.5% (w/v) NMS or chitosan (Sigma-Aldrich, USA) and 0.05% cysteine-HCl (Sigma-Aldrich, USA) at pH 6.0 for 48 h at 37 °C (Baker Ruskinn, UK). Bacteria were introduced into each media type in triplicate with 1 in 100 inoculation. To track bacterial growth in each media type, 10 µl samples from one replicate (at T = 0 h) or all three replicates (at T = 48 h) of each media type and strain were removed for tenfold serial dilution (10⁻²–10^–7) and plating on MRS plates. After 48 h anaerobic incubation as above, colonies formed were counted and colony forming units per ml (CFU/ml) were calculated.