We observed that yeast transformants displaying mCherry showed colour change on YNB-casamino selective plates and culturing medium.
A second SiO2 layer was deposited via an HDP CVD process in order to electrically isolate the entire electrode from buffer solution and protect the electrodes from the chemical reaction in the wet cell culturing medium.
Immunocompetent male Brown Norway rats (Harlan Laboratories, Horst, the Netherlands), with a body weight of approximately 250 g, were inoculated with 3 × 105 BN7005-H1D2 cells in 50 μL culturing medium between the peritoneum and the abdominal muscle under anesthesia with isoflurane (Abbott Scandinavia AB, Solna, Sweden).
All substances were obtained from Sigma Aldrich and dissolved in culturing medium.
An excess of culturing medium was used to quench the trypsinization reaction.
The following day, transfected VMN-Wnt5a spheres were collected by gentle scraping, spun down at 800 RPM, and washed once in culturing medium before being transferred to new culturing medium.
Prior to in vitro grafting, VMN or VMN-Wnt5a cells were isolated, spun down at 800 RPM for 5 min and each (6 cm diameter) culturing dish re-suspended in 30 40 µl of organotypic slice culturing medium.
The slices were separated and transferred to sterile, porous membrane units (0.4 µm, Millicell-CM, Millipore, Temecula, CA), five per insert, containing 1 ml of culturing medium in each dish (50% MEM with Earle's salt and L-glutamine, 25% HBSS, 25% horse serum with 6.5 mg/L D-glucose, 20 mM HEPES and 50 U/ml streptomycin-penicillin).
After slicing, sections were kept 15 min in ice-cold washing medium containing HBSS with HEPES 20 mM, glucose 17.5 mM, NaOH 0.88 mM and penicillin/streptomycin (all from Gibco) before placing individual slices on membrane inserts (Millipore PICM01250) in 240 µl culturing medium in 24-well dishes.
Each MEA culturing well contained 1.5 ml of culturing medium.
Antibodies were purified from collected hybridoma culturing medium by affinity chromatography using immobilized antigen peptide.
