The growth of cells in culture follows a standard pattern. A leg after seeding is followed by a period of exponential growth called the log phase. Cells should be passaged when they cover the plate or the cell density exceeds the capacity of the medium. Maintaining log phase growth will maximize the number of healthy cells for your experiment. Before retrieving your flasks from the incubator, sterilize the hood and have all required supplies at hand. Examine your culture carefully for signs of contamination or deterioration. Handle the cells with care during transport. For adherent cells, remove the spent medium from the flask using a sterile pipette. Rinse the cells with a balanced salt solution, such as DPPS. Make sure to use a salt solution without calcium and magnesium, as these may inhibit your cell dissociation reagent. After rinsing the cells, remove the salt solution. Each time you aspirate liquid off the cells, place the next solution on quickly. Add your cell dissociation reagent to remove cells from the plate. Use just enough solution to cover the cell sheet. Consider using a gentle dissociation reagent, such as TrypLE Express, to avoid damaging your cells during dissociation. Make sure that the solution completely covers the cells. You may want to tap gently on the plate to help the cells detach. Use a microscope to confirm the cells have released from the flask. They will start to appear round as they release from the substrate and will move or slide when the flask is tilted. Complete dissociation is necessary. Do not leave the dissociation reagent on too long, especially if you’re using a reagent other than TrypLE. You may choose to manually break up lingering clumps by repeatedly pipetting warmed medium over them. A single-cell suspension is important to achieve an accurate cell count. If you are using trypsin, the collection medium will need serum or you’ll need to use a trypsin inhibitor to inactivate the trypsin. If you’re using TrypLE, inactivation is achieved by dilution alone. No serum is needed. Transfer the cells to a conical tube and centrifuge to remove any residual dissociation reagent. The speed and time of centrifugation will vary based on your cell type. After centrifugation, you should have a well-formed pellet. Remove the medium from the centrifuge tube with a pipette and discard the medium into a waste container. Try not to disturb the pellet. Resuspend the pellet with warm complete growth medium. Gentle pipetting will disperse the cells to ensure a homogeneous solution of single cells. Remove a small sample for cell counting. Trypan blue is used when counting cells to indicate the ratio of live to dead cells. The stain turns dead cells blue, but healthy cells with an intact membrane will remain white or colorless. Using the microscope, count the total number of cells and the number of dead or blue cells. Based on your cell count, determine how much additional fresh medium to add for optimal seeding density. Add the required medium, mix the cells gently, and pipette the solution into fresh flasks. Cap the vented caps on your flask tightly. If the caps are not vented, cap loosely. This keeps contamination out while allowing adequate gas exchange. Use a north, south, east, west motion to evenly distribute the cells and transport the flasks to the incubator. When passaging suspension cells, you’ll begin by removing a small sample from the cell culture flask for counting. You’ll follow the same counting procedure for adherent cells, using trypan blue and either a hemocytometer or the Countess automated cell counter. Based on your cell count, add additional fresh medium to the flasks. Stay within the minimum and maximum volumes. This is important to maintain optimal air exchange and shaking flow. You may need to split the culture into multiple flasks. Cap the flasks appropriately, depending upon whether they are vented or not, and return them to the incubator. Every three weeks, gently spin the cells, remove the medium, and replace with completely new medium. This eliminates cell debris and metabolic waste.