Antibiotic antimycotic solution 100x

We tested tissue samples (lung, liver, spleen, heart, kidney, and placenta, if delivered) from 64 aborted fetuses that were delivered to the Department of Virology of the National Veterinary Research Institute in Pulawy between 1999 and 2012. The whole fetuses or fetal organs came from horse studs located in different regions of Poland. None of the animals had been vaccinated against EHV-1, and none of the studs had a history of respiratory and neurological disease. Necropsy reports revealed that all 64 abortions occurred during the third trimester of pregnancy. A variety of macroscopic lesions were observed in most cases, including a large amount of pleural fluid, hepatic necrosis, and pulmonary oedema. No histological investigation was done. Organ samples from aborted fetuses were stored at −70°C until further processing.
Two grams of each tissue sample was used for preparation of 10% (w/v) suspension in Eagle’s Minimum Essential Medium (Sigma-Aldrich), supplemented with 1% antibiotic solution (Antibiotic Antimycotic Solution 100x, Sigma-Aldrich) using ULTRA-TURRAX® homogenizer. Tissue homogenates were centrifuged at 1,700 x g for 10 min, and then supernatants from the same fetus were pooled together and stored at −70°C until testing.

Three human tumors lines: HT29 (colon adenocarcinoma, ATCC HTB-38), HCT116 (colon carcinoma, ATCC CCL-247), DU145 (prostate carcinoma, ATCC HTB-81), and normal human skin fibroblast cell line BJ (ATCC CRL-2522) were cultivated according to the depositor’s proposed protocols (ATCC, American Tissue and Cell Collection) at 37°C and 5% CO₂ in DMEM culture medium (Gibco, ThermoFisher Scientific, Waltham, MA, US) supplemented with 10% FBS (Euroclone, Milan, Italy), and a stabilized Antibiotic Antimycotic Solution (100x) (Sigma, Saint Louis, MO, US) containing 10,000 units penicillin, 10 mg streptomycin and 25 μg Amphotericin B per mL. This culture medium will be further designated as complete culture medium.

NaCl, CaCl₂, MgCl₂, tricine, DMSO, and antibiotic antimycotic solution (100X) were purchased from Sigma-Aldrich (Saint Louis, MO). NaOH and KCl were from EMD Chemicals (San Diego, CA). Glucose (dextrose), RPMI 1640, and gentamicin sulfate were from Thermo Fisher Scientific (Waltham, MA). Fura PE3 acetoxymethyl (AM) ester from Cayman Chemical (Ann Arbor, MI) was the Ca²⁺ reporter used. Pluronic F-127 was from Life Technologies (Grand Island, NY). Collagenase P (from Clostrdium histolyticum) was acquired from Roche Diagnostics (Indianapolis, IN). Cosmic Calf Serum was purchased from GE Healthcare Bio-Sciences (Pittsburgh, PA). Poly(dimethylsiloxane) (PDMS) base and curing agent were procured from Dow Corning (Midland, MI, USA). SU-8 2075 photoresist was from Microchem (Westborough, MA). All solutions were made with Milli-Q (Millipore, Bedford, MA, USA) 18 MΩ·cm ultrapure water. Glucose solutions were prepared with a buffered salt solution composed of 2.4 mM CaCl₂, 125 mM NaCl, 1.2 mM MgCl₂, 5.9 mM KCl, and 25 mM tricine.

Immortalised RAW264.7 macrophages (400319, CLS Cell Lines Service) were cultured in Dulbecco's modified Eagle's medium-high glucose (DMEM, D5671, Sigma-Aldrich), enriched with 10% fetal calf serum (FCS, P30-3302, PAN Biotech), 1% L-glutamine (G7513, Sigma-Aldrich), and 1% Antibiotic/Antimycotic Solution 100x (A5955, Sigma-Aldrich). The medium was replaced every two to three days. Cultivation was performed in an incubator (BBD 6220, Thermo Fisher Scientific) at 37°C and 5% CO₂ saturation. All cell culture experiments were carried out under sterile conditions (laminar flow unit, BDK Luft- und Reinraumtechnik).

Laboratory-grade hyaluronic acid (HA) sodium salt (1500–1750 kDa) was purchased from Contipro a.s. (Dolní Dobrouč, Czech Republic). Sulfo-Dibenzocyclooctyne-PEG₄-amine (Sulfo DBCO-PEG4-NH2) was bought from Click Chemistry Tools (Scottsdale, AZ). N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), azide-terminated poly(N-isopropylacrylamide) (pNIPAM-N₃; 15 kDa), hyaluronidase from bovine testes (Type VI-S), hydrogen peroxide (30% w/w), toluidine blue, Dulbecco’s Modified Eagle’s Medium—high glucose (DMEM), embryoMax L-glutamine solution (100X), foetal bovine serum (FBS), and the antibiotic–antimycotic solution (100X) were purchased from Sigma-Aldrich (St Louis, MO, USA). Dialysis membranes (Biotech CE Dialysis Tubing 300 kDa) were acquired from Repligen (Waltham, MA, USA). The Alcian blue PAS stain kit was purchased from Abcam (Cambridge, UK). Belotero Balance^® and Belotero Volume^® were purchased from Merz Pharma (Geneva, Switzerland) and used as reference products.