Cells were plated and grown as previously described to produce primarily Intermediate architectures [8 (link)]. After 24 hrs, media was removed and cells were washed with PBS, after which cells were incubated for the specified time in magnesium-calcium-free PBS supplemented with 2 mM Magnesium dichloride. We found that for treatments of up to 3 hours in calcium free media, cell monolayers remained intact and mostly confluent, though there were occasionally cell scale gaps that would develop in the monolayer. These small gaps are unlikely to affect classification by ALAn and are unlikely to impact average architecture of the tissue.
For blebbistatin treated monolayers, cells were grown to an Intermediate architecture and treated with 50 μg/ml of blebbistatin or the equivalent amount of DMSO as a control for the specified time. After treatments cells were fixed and stained as per the above protocol.
For blebbistatin treated monolayers, cells were grown to an Intermediate architecture and treated with 50 μg/ml of blebbistatin or the equivalent amount of DMSO as a control for the specified time. After treatments cells were fixed and stained as per the above protocol.
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