Seaplaque gtg

To avoid cell attachment to the plate bottom and to stimulate 3D cell culture generation, 96-well f-bottom plates (Greiner Bio-one, Frickenhausen, Germany) were coated with pre-warmed SeaPlaque® GTG (Cambrex Bio Science Rockland, Rockland, ME) agarose or with MC solution as described below. For the OM1 subgroup the MC 1,5% solution was prepared as described for the IM method, but FCS was not included. For the OM2 subgroup, sea plaque agarose (SPA) was diluted in RPMI 1640 medium with l-glutamine without FCS to a final concentration of 1.5% and then autoclaved together with a magnetic stirrer. Thereafter, for the OM1 subgroup 96-well f-bottom plates (Greiner Bio-one, Frickenhausen, Germany) were coated with 50 μl per well of pre-warmed 1.5% MC solution. For the OM2 subgroup 96-well f-bottom plates (Greiner Bio-one, Frickenhausen, Germany) were coated with 50 μl per well of pre-warmed 1.5% SPA solution, respectively. After the first layer had been allowed to solidify, a single-cell suspension containing 10⁴ cells per 150 μl was plated in complete growth medium into each well. The plates were centrifuged at 800 rpm for 15 min to allow cell-to-cell contact and incubated in a humidified atmosphere at 37°C and 5% CO₂. The 3D cell cultures were observed for 24 DiV of culturing and medium was changed every other day by replacing 50% of the medium.

Genomic DNA was isolated from 16 days old culture in log phase using the Himedia Ultrasensitive Spin Purification Kit (MB505) following the instructions of the manufacturer, except for the increase of incubation time for the lysis solutions Al and C1, which were set to 60 and 20 min, respectively. DNA fragments within the following genes were amplified using the oligonucleotide primers and PCR programs listed in Table S1. PCR products were checked by electrophoresis on 1% agarose gels (SeaPlaque® GTG®, Cambrex Corporation), using standard protocols. The products were purified directly using the Geneclean® Turbo kit (Qbiogene, MP Biomedicals) and sequenced using the BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems, Life Technologies). The partial sequences were compared with the ones available in the NCBI database using BLASTN. The BLAST X tool (blast.ncbi.nlm.nih.gov/Blast.cgi) was used for determination of the nos, mcy G and mcy D genes similarity. The sequences were annotated for the coding regions by the NCBI ORF Finder at NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and the ExPASY proteomics server (http://www.expasy.org/tools/dna.html).