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A subconfluent T25-flask typically contains between 0.9 and 1.2 million cells corresponding to 36;000-48;000 cells per cm2. It is recommended to count the existing cell number after trypsinization and to calculate the needed number of new flasks. Recommended seeding density for HUVEC is 5;000-10;000 cells/cm2. This usually corresponds to a split ratio of 1:4-1:6. 1:6 means that you can increase the culture surface by factor 6 (e.g. from 1x T25 to 6x T25 or 2x T75).