Trypsin ethylenediaminetetraacetic acid

Ethical approval for the collection of bone marrow aspirates and blood was received from the Ethics and Welfare Committee at the Royal Veterinary College (URN 2013 1230R 2005). No horses were euthanased for the sole purpose of obtaining tissues for this study. Bone marrow-derived MSCs (n = 3) were obtained and expanded as described previously [9 (link)] and P0 to P2 passage cells were stored in Bambanker™ cell freezing medium (Anachem, Luton, UK) in liquid nitrogen until use. For experiments, cells were seeded in D10 medium (Dulbecco’s modified Eagle’s medium, supplemented with foetal calf serum (10% v/v), 100 U/ml penicillin, and 100 U/ml streptomycin; all from Invitrogen, Paisley, UK) and were expanded to the required numbers. Cells were then detached from the culture flasks by trypsin–ethylenediamine tetraacetic acid (Sigma-Aldrich, Gillingham, UK) for storage media and needle gauge experiments.

Six male intermittent catheters
of CH12 size were evaluated: four hydrophilic PVP-coated catheters
(Coloplast SpeediCath Flex, Coloplast Luja, Hollister VaPro Pocket
and Wellspect LoFric Origo), an IAS catheter (GentleCath Glide with
FeelClean Technology-Convatec Ltd., Deeside, UK), and an uncoated
catheter (Self-Cath-Coloplast Ltd., Peterborough, UK). T24 bladder
carcinoma cells [LCL-1709, American Type Culture Collection (ATCC)
HTB-4] were purchased from ATCC (Virginia, US). McCoy’s medium,
penicillin–streptomycin (10,000 U/mL), Trypsin-ethylenediaminetetraacetic
acid (0.5%), 10% fetal bovine serum (FBS), toluidine blue-O (>80%),
phosphate-buffered saline (PBS), paraformaldehyde (>99%), and fibronectin
(>95%) were obtained from Sigma-Aldrich (Ireland Limited, Wicklow,
Ireland). Hoechst stain (2 μg/mL 33258, bisbenzimide) was purchased
from Abcam (Cambridge, UK). Red food coloring was bought from a high-street
store.

Tissue specimens were mechanically dissociated using a scalpel and transferred to a solution of 20 mg/mL collagenase I (Gibco BRL Co., Invitrogen) in DMEM medium and incubated at 37 °C for 15 h on a shaking incubator (Grant Bio, Keison Products, UK). Specimens dissociated into single cells were washed with 10× excess of phosphate-buffered saline (PBS) and separated cells were collected by centrifugation at 300×g. Cells were plated in IMDM with 10% FBS and, after cell adhesion, 10 μM Rho-associated protein kinase (ROCK) inhibitor was added to the culture medium for 1 h and the medium in the plates was replaced with fresh complete IMDM medium. At the next passages, cells were cultured in complete IMDM medium supplemented with epithelial cell growth supplement (#6622, Cell Biologics, Chicago, IL, USA), Mito + Serum Extender (BD Biosciences - Discovery Labware, San Jose, CA, USA), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 mg/mL amphotericin B and were cultivated in 6-well plates at 37 °C in a humidified atmosphere containing 5% CO₂. When 70–80% confluence was reached, cells were harvested using 0.05% trypsin/ethylenediaminetetraacetic acid (Sigma-Aldrich) and sub-cultured for further experiments.

Ammonium acetate, acetone, and 2-propanol and formic acid werepurchased from Carlo Erba (Milan, Italy). Standard solutions of vanillic acid, syringic acid and chlorogenic acid were purchased from Extrasynthese (Genay Cedex, France). Acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). The solid-phase extraction SPE cartridges used were Waters Oasis Hydrophilic-Lipophilic Balance (HLB) (200 mg) and ultrapure deionized water, with resistivity of 18.2 MΩ cm, obtained from the Milli-Q^® Integral water purification system with Q-pod (Millipore, Bedford, MA, USA). All solutions were filtered through 0.45 membranes (Millipore, Bedford, MA, USA) and degassed before use. Ferulic acid, gallic acid, kaempferol, quercetin, and rutin, N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% of trimethylchlorosilane, Di(2-ethylhexyl)phthalate, Dulbecco’s modified eagles Medium (DMEM) high glucose, 2′,7′-dichlorodihydrofluoresceine diacetate (DCFH2-DA), 3-4,5-dimethylthiazol-2-yl,2,5-diphenyltetrazolium bromide (MTT), phosphate buffer saline (PBS), fetal bovine serum (FBS), streptomycin and penicillin mixture, and trypsin-Ethylenediaminetetraacetic acid (EDTA) were acquired from Sigma-Aldrich S.r.l. (Milan, Italy).

Dulbecco′s Modified Eagle Medium (DMEM) high glucose, trypsin-ethylenediaminetetraacetic acid, trypan blue solution 0.4% (w/v), sodium dodecyl sulphate, hydrochloric acid (HCl), sodium bicarbonate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red (NR) solution, Hoescht 33258 solution, dimethyl sulfoxide, retinoic acid, paraformaldehyde, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), ACRO, and FBAL were obtained from Sigma-Aldrich (Taufkirchen, Germany). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbo-cyanine iodide (JC-1) was obtained from Thermo Fisher Scientific (Waltham MA, USA). Phosphate-buffered saline (PBS) without calcium and magnesium and penicillin/streptomycin were obtained from Biochrom (Berlin, Germany). Foetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS) were acquired to Gibco (Paisley, UK). Doxorubicinol hydrochloride was obtained from Tebu-bio (Lisbon, Portugal). All plastic sterile material used in cell culture was obtained from Corning-Costar (Corning, NY, USA).