50 ml centrifuge tube

Collagen type I was isolated from rat tail tendons in a 1N HCl solution (Fisher Scientific, Hampton, NH, USA) at an approximate pH of 2.0 and was stirred for 16 h at room temperature, as published previously [28 (link),38 (link),40 (link),41 (link),42 (link),43 (link)]. The solution was then transferred to 50 mL centrifuge tubes (Fisher Scientific, Hampton, NH, USA) and centrifuged at 30,000× g (16,000 rpm) for 60 min at 4 °C to pellet the insoluble components. The supernatant was decanted from each tube and transferred into separate 50 mL centrifuge tubes, stored at −20 °C overnight, and freeze-dried for 48 h. Samples were dissolved in an appropriate amount of 0.01% glacial acetic acid (Fisher Scientific, Hampton, NH, USA) for a final concentration of 8 mg/mL of collagen stock solution.

Collagen type I was isolated from rat tail tendons donated at the conclusion of other studies at the University of Texas Austin (IACUC Protocol ID: AUP-2022-00040). Rat tail tendons were placed in a 1 N HCl solution (Fisher Scientific, Hampton, NH, USA) at approximately at pH 2.0 and stirred for 16 h at room temperature. The solution was then transferred to 50 mL centrifuge tubes (Fisher Scientific, Hampton, NH, USA) and centrifuged at 30,000× g (16,000 rpm) for 60 min at 4 °C to pellet the insoluble components. The supernatant was decanted from each tube and transferred into separate 50 mL centrifuge tubes, stored at −20 °C overnight, and freeze dried for 48 h. Samples were dissolved in an appropriate amount of 0.01% glacial acetic acid (Fisher Scientific, Hampton, NH, USA) for a final concentration of 8 mg/mL of stock solution [30 (link)].

Collagen type I was isolated from rat tail tendons in a 1 N HCl solution (Fisher Scientific, Hampton, NH) at approximate pH 2.0 and stirred for 16 h at room temperature. The solution was then transferred to 50 ml centrifuge tubes (Fisher Scientific, Hampton, NH) and centrifuged at 30,000×g (16,000 rpm) for 60 min at 4 °C to pellet the insoluble components. The supernatant was decanted from each tube and transferred into separate 50 ml centrifuge tubes, stored at −20 °C overnight, and freeze-dried for 48 h. Samples were dissolved in the appropriate amount of 0.01% glacial acetic acid (Fisher Scientific, Hampton, NH) for a final concentration of 6 or 14 mg/ml of collagen stock solution [64 ] to create 3 and 7 mg/ml collagen hydrogels, respectively.

Cultures were harvested just prior to medium nitrogen depletion and at the conclusion of each experiment. Each time, two aliquots of 50 mL were dispensed into 50-mL centrifuge tubes (Fisher Scientific, Palatine, IL) and centrifuged (Thermo Scientific, Sorvall Legend XTR, Waltham, MA) at 4800×g at 4 °C for 10 min. The concentrated biomass was rinsed with diH₂O to remove media salts and excess bicarbonate, before centrifuging again. The remaining algal pellets were rapidly frozen and lyophilized (Labconco lyophilizer, Kansas City, MO) for 48 h and stored at −20 °C for subsequent lipid analysis.

Zinc diethyldithiocarbamate (Zn (DDC)₂) (molecular weight of 361.99 g/mole and >99.0% purity) was purchased from Tokyo Chemical Industry (TCI) Co., Ltd., Tokyo, Japan. 2-hydroxypropyl-beta-cyclodextrin of USP grade (HP) (molecular weight: 1555 g/mole) and Sulfobutyl ether beta-cyclodextrin sodium (SBE) (molecular weight: 2242.05 g/mole) were bought from Glentham Wiltshire, UK. MD. Acetonitrile laboratory of reagent grade > 99% (molecular weight: 41.05 g/mole) was purchased from Fisher Scientific, Loughborough, UK. Lung cancer cell lines A549 were obtained from ATCC (Teddington, UK). Dulbecco’s modified Eagle’s medium (DMEM), Gibco™ foetal bovine serum (FBS), penicillin streptomycin (Pen-Strep) antibiotic solution, non-essential amino acid solution and L-glutamine (cell culture-tested, 99.0–101.0%), 50 mL centrifuge tubes (sterile), tissue culture flask of 75 cm² (sterile), serological pipettes (sterile), and Gibco™ Trypsin-EDTA (0.25%), were purchased from Fisher Scientific, Loughborough, UK. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Dimethyl sulfoxide (DMSO) were purchased from Glentham Life Science, Wiltshire, UK. Corning^® 96-well TC-treated microplates were obtained from Sigma-Aldrich, Dorset, UK.