Gibco dulbecco s modified eagle medium

FLSs were isolated from the rats in the AA model group as described previously (24 (link)). Sterile synovial tissue samples were separated into 1-mm³-sized pieces, mixed with twice the volume of 0.2% type II collagenase (Merck KGaA) containing 10% Gibco^® fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the tissues were digested for 2–2.5 h at 25°C (percussing the samples once every 30 min). Finally, 0.25% trypsin was added for a further digestion step for 30 min, and the isolated FLSs were cultured in Gibco^® Dulbecco's modified Eagle medium (Thermo Fisher Scientific, Inc.) supplemented with 15% FBS in an incubator at 37°C with 5% CO₂ prior to performing the following experiments.

rAAV vector production was performed as described previously.²³ (link)^–25 (link) Briefly, HEK293T cells were triple transfected with an rAAV helper plasmid (pHelper), a self-complementary (sc) green fluorescent protein (GFP) reporter plasmid with a ubiquitously expressing chicken beta-actin promoter (CBA-scGFP), and an AAV2-based rep-cap plasmid—for either unmodified AAV2 rep-cap plasmid (pACG2) or a mutant rep-cap plasmid encompassing one of seven candidate sequences (pACG2-Peri-A, pACG2-Peri-B, pACG2-Peri-C, pACG2-Peri-D, pACG2-Peri-E, pACG2-Peri-F, and pACG2-Peri-G). Equimolar ratios of plasmids were transfected into HEK293T cells and grown in high-glucose Gibco Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum and 1% antibiotic–antimycotic (Thermo Fisher Scientific, Waltham, MA). rAAV vector purification was performed 72 hours post-transfection by iodixanol density gradient centrifugation and buffer exchange using 100-kDa columns (Amicon, Darmstadt, Germany). Finally, rAAV was washed and eluted in Hank's balanced salt solution containing 0.014% Tween 20, and the titer was calculated using a PicoGreen assay (Thermo Fisher Scientific).²⁴ (link)

Lung cancer (A549) and neuroglioma (H4) cell lines were obtained from ECACC (Wiltshire, UK). A549 cell line was grown in Gibco RPMI media 1640 (ThermoFisher, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (ThermoFisher, Paisley, UK), while H4 cell line was cultured in Gibco Dulbecco's Modified Eagle Medium (ThermoFisher, Paisley, UK) supplemented with 10% FBS and 1% penicillin–streptomycin. A549 and H4 cells were cultured at 37 °C with 5% CO₂ and balanced with air. Cells were treated with 4 μg/mL propofol (Sigma-Aldrich, Dorset, UK) for 2 h when the seeded cells formed a continuous monolayer. Intralipid (Santa Cruz Biotechnology, Dallas, Texas, USA) was used as vehicle control. After which, cell media was replaced with fresh media for the next 24 h for further experiments.

Crosslinked AMs and normal AMs were placed in 6-well plates; 1 ml of serum-free Gibco Dulbecco's modified Eagle medium (DMEM; Thermo Fisher Scientific) was then added to each well. Each piece was 9 cm² in size. The supernatants incubated for 24 hours in serum-free medium were obtained to determine the protein content of the supernatants and the growth factors present in each sample. The analysis of growth factor levels was carried out using a Quantibody Human Growth Factor Array kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer's protocols. Briefly, after a 30-minute incubation at room temperature with sample diluent, the glass chips were washed five times, and each well arrayed with human growth factor antibodies was overlaid with 100 µl of medium. After overnight incubation at 4°C and extensive washing, the detector antibody was added for 1 hour and then washed away, and Alexa Fluor 488 conjugated streptavidin was added for 2 hours at room temperature. The signals were scanned with a fluorescence laser scanner. Each sample was prepared in quadruplicate. Growth factors were quantified with the Q-Analyzer tool (#QAH-GF-1-SW; RayBiotech) against a standard curve set for each growth factor, with a five-point serial dilution of growth factor standards.

Mouse neuroblastoma x spinal cord (NSC-34) cells (a gift from Prof. Neil Cashman, University of British Columbia, Vancouver) were cultured in Gibco Dulbecco's Modified Eagle Medium (Life Technologies, 10313) with 10% (v:v) heat inactivated fetal bovine serum (Sigma-Aldrich, 12003C), 1% (v:v) Gibco penicillin-streptomycin (Life Technologies, 15140) and 1% (v:v) Gibco glutamine (Life Technologies, 25030). pEGFP-N1 vector (Clontech, 6085-1) containing human wild-type (WT) or mutant (A4V, G85R, G93A) SOD1 cDNAs with a C-terminal EGFP tag were generated as previously described [39 (link)]. pmCherry-N1 vector (Clontech, 632523) containing human WT or mutant (Q331K, M337V) TARDBP cDNAs with a C-terminal mCherry tag were generated as previously reported [54 (link)]. Stable cell lines expressing SOD1-EGFP or TARDBP-mCherry constructs were generated as previously described [54 (link)].