Hello Dainius Characiejus

Cell clumps form in culture because of the presence of free DNA in cell suspension which are released from dead cells. Free DNA attract cells which bind altogether forming clumps.

Dead cells are formed when you usually subculture over-confluent cells. Also, over exposure of cells to trypsin during trypsinization can lead to cell death. So, subculture cells when they are 70-80% confluent. Also, during trypsinization expose cells to trypsin for not more than 5 mins.

If the clumps are small, you may pipette the cell suspension a few times gently up and down to form single cell suspension. If you still do not succeed, you may consider using DNase I at concentrations ranging from 20-100µg/ml to avoid formation of clumps. For each cell type, the working concentration must be determined individually.

Best.

Cell clumping can occur for several reasons, and it is not necessarily indicative of a mistake in your methodology. It is important to note that some cells are naturally more prone to clumping than others. In the case of B16 melanoma cells, they tend to clump together due to their nature.

Here are a few possible reasons why cell clumps were formed:

In summary, some cells are naturally prone to clumping, and several factors can contribute to the formation of cell clumps. You may try optimizing the trypsinization conditions, including time and concentration, as well as the technique used to prepare the cell suspension.

Hello Yali 陈雅黎 Chen , thank you very much for your very thorough explanation. This is really helpful!

You're welcome, glad this helped you