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The optimized complex and defined media were subsequently validated and compared with the original malt extract broth or MEB.
Basolateral media were subsequently collected and cells were rinsed twice in ice-cold PBS and lysed in a buffer consisting of 10 mM Tris, 150 mM NaCl, 0.025% sodium azide, 5 mM EDTA, 0.1% SDS, 1% Triton X100 and 0.5% sodium desoxycholate together with 1 mM of each antiprotease: pepstatin A, PMSF and aprotinin.
Solid media were subsequently incubated for 48 h at 36°C with 5% CO2.
The cells were incubated for 4 h, and the media were subsequently changed to antibiotic- and serum-containing MEM.
OVSAYO and OVISE cells were cultured under normoxia or 1% O2, and conditioned media were subsequently collected.
The media were subsequently aspirated and the cells were treated with 30 or 60 μM C-6 or the corresponding 0.06% (v/v) DMSO vehicle control.
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The supernatant obtained was filtered through 0.22 µm filter to remove any bacteria left behind and the filtrate (conditioning media) was subsequently inoculated with 106 cells of PP9 and PP9: E. coli JM 101 (log ratio 1∶1).
This culture media was subsequently added to cultures of pericytes.
The media was subsequently changed every other day.
Cell media was subsequently removed and cells were rinsed with PBS twice.
Lipofectamine-containing media was subsequently removed and replaced with standard growth media (DMEM-F12 containing 5% FBS) for further 24 h before treating as appropriate for individual experiments.
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