Where samples were stained with crystal violet, supernatants containing MTS reagent were carefully aspirated and the remaining cell monolayer was fixed in 4% buffered PFA solution for 10 min. The monolayer was then stained in 0.1% crystal violet solution for 30 min, washed with water, and air-dried. Wells were observed under a Zeiss Axiovert 25 Inverted Light/fluorescence Microscope (Zeiss), the images recorded using a Nikon DS-U2 camera (Nikon), and processed using NIS-element AR 3.00 software.
Where samples were obtained for qPCR analysis, additional wells were seeded and harvested before the addition of MTS reagent. Supernatants were removed, the monolayer gently washed with 100 μL 10% FCS in DMEM, and the wash pooled with the supernatant fraction before centrifugation at 300 g. The supernatant fraction was then collected before snap freezing in liquid nitrogen and storing at −80°C. The cell fraction was obtained by adding trypsin to the monolayer before adding 100 μL 10% FCS in DMEM and centrifuging at 300 g. The supernatant was discarded and the pellet resuspended in 200 μL 10% FCS in DMEM. The cell fraction was then combined with any residual cells removed by centrifugation from the supernatant fraction.
Where samples were obtained for qPCR analysis, additional wells were seeded and harvested before the addition of MTS reagent. Supernatants were removed, the monolayer gently washed with 100 μL 10% FCS in DMEM, and the wash pooled with the supernatant fraction before centrifugation at 300 g. The supernatant fraction was then collected before snap freezing in liquid nitrogen and storing at −80°C. The cell fraction was obtained by adding trypsin to the monolayer before adding 100 μL 10% FCS in DMEM and centrifuging at 300 g. The supernatant was discarded and the pellet resuspended in 200 μL 10% FCS in DMEM. The cell fraction was then combined with any residual cells removed by centrifugation from the supernatant fraction.