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The QIAGEN Guide to Animal Cell Culture

it ratio required (see step 2) and the size of the cell culture vessel. If too small a volume is used, it may be difficult to accurately pipet the desired volume to the new culture vessel. Conversely, if too large a volume is used, the culture vessel may be too full following transfer of the cells.

Tip - Removal of trypsin may sometimes be necessary following harvesting of adherent cells, especially for primary and sensitive cell types. Centrifuge the cells at 200 x g for 5 min, carefully aspirate the supernatant, and resuspend the cells in an appropriate volume of prewarmed medium containing serum.

2. Transfer an appropriate volume of the resuspended cells to a fresh cell culture vessel containing prewarmed growth medium. Swirl the vessel gently to mix the cells with the medium.

IMPORTANT: Thoroughly mix the cells in the cell culture vessel to ensure even distribution of cells.

IMPORTANT: Some cell types will not survive if too few cells are transferred. We do not recommend high split ratios for primary cells, sensitive cell types, or senescent cultures.

Tip - For adherent cells, we recommend adding enough cells so that the culture takes approximately one week to reach confluence again. This minimizes the number of times the cells are trypsinized as well as the handling time required to maintain the culture.

Tip - When determining how many cells to transfer to the new cell culture vessel, it can be helpful to think in terms of how many cell divisions will be required for the culture to reach confluence again. For example, if half the cells are transferred, t
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