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Passaging is required every week in order to prevent cell senescence due to contact inhibition. Here, we demonstrate a common procedure of passaging adherent cells to obtain new flask concentrations of 2.5 x 10^5 cells/mL Here is the procedure: 1. Assess confluence 2. Remove media 3. Add 2 mL trypsin for 45 seconds, then remove 1 mL 4. Agitate to get all cells suspended 5. Neutralize with 2.5 mL DMEM +10% CS 6. Obtain 0.01 mL suspended cells, and mix with 0.01 mL typhan blue. 7. Load onto a hemocytometer slide and record the cell concentration. 8. Determine the volume needed for each flask to have 2.5 x 10^5 cells/ mL
