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After filling the lower chamber with complete media, 2 × 104 HCC cells in 0.5 mL serum-free media were loaded onto the upper chamber.
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Interestingly, the origin of Xin ca. 550 million years ago coincides with the genesis of heart chambers with complete endothelial and myocardial layers.
The heart in zebrafish and lamprey are similar in organization and consist of two chambers with complete endothelial and myocardial layers which are also hallmark features of the heart in all later-diverging taxa [30], [31].
Then 5 × 10 cells were added to the upper chamber, whereas lower chamber were filled with complete medium.
Cells were then re-suspended in DMEM and were added to the upper chamber while the lower chamber was filled with complete media as a chemoattractant.
Cells (2 × 10) suspended in 100 μ l serum-free medium were added to the upper chamber and the lower chamber was filled with complete medium with 10%% serum.
After 16 hours, the lower chamber was replaced with complete media and the upper chamber replaced with serum starve media.
The bottom chamber was filled with complete growth medium containing chemoattractant growth factors.
HUVEC grown in complete media were seeded in triplicate in 8-well chamber slides coated with complete Matrigel (4×104 cells/well).
For RAW 264.7 chemotaxis under hypoxic conditions, the lower chamber was supplemented with complete DMEM containing 10% FBS to create a chemotactic gradient.
HUVEC seeded in serum-free media in 8-well chamber slides coated with complete Matrigel (4×104 cells/well) were allowed to adhere before being sandwiched with another layer of Matrigel that was uniformly pipetted on top of the adherent cells.
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