Subculturing monolayer cultures is a routine task in every cell culture lab. To minimize the impact on cell culture quality and assay outcome, the procedure should be standardized wherever possible. While some variables,such as culture medium, dissociation agent or cell culture vessel are easy to mitigate, the time point of subculture and the dissociation itself remain user dependent.
The CyoSMARTTM System with its growth curve recording and automatic alert function enables you to always subculture your cells at the same cell density and avoid subjective determination of confluency. Cells should be in log phase (exponential growth phase) when subcultured, ideally at 70 to 80% confluency.
The detachment of the cells from the cell culture vessel is one of the most important steps in the subculturing process and is, unfortunately hard to standardize. When using an enzymatic dissociation agent, the temperature and the freshness have an important impact on the enzymatic activity of the enzyme and thus on the detachment. Ideally the enzyme should be aliquoted and for each subculture a fresh aliquot should be used. Depending on how strongly cells attach to their culture vessel surface, the dissociation can be performed at room temperature or in an incubator. In any case, it is important to monitor the dissociation under a microscope to avoid damage to the cells. This observation is easily done when cells are incubated with the enzyme at room temperature. Use of the CytoSMARTTM System means that close observation is possible even in the incubator. Put your cell culture with the enzymatic agent on the CytoSMARTTM System in the incubator and observe the cells lifting from the culture vessel from the tablet. The ideal time point to stop the enzymatic activity by adding serum containing media, PBS/BSA, Trypsin inhibitor, or Trypsin Neutralization Solution is when the cells are rounded up and only a few cells start to float.