Hi,
I'm trying to stain my cells after transient transfecting them but each time I get the cells either over confluent, or clumped in the middle even though I swirl the dish in an 8 shape to prevent clumping. any ideas would be appreciated it
Thank you
Hanging cells may hang together and form blocks for several reasons. The most common cause of cell aggregation is the presence of free DNA and cell debris in the center of culture, which occurs after cell decomposition. The viscous nature of the DNA in the cell pool and other debris causes large clumps. Here are some causes of cell decomposition and DNA release in culture media.
1. Hyperglycemia: Excessive treatment by protein-soluble enzymes - such as trypsin, commonly used to separate cells - can lead to cell aggregation
2. Environmental stress: Mechanical strength, repeated freezing / melting cycles and other environmental stresses can accelerate cell death, an early symptom of cellular cell adhesion.
3. Tissue Classification: Preparation of mono-cell suspension from primary tissue through chemical, mechanical or enzymatic protocols can lead to cell rupture. Collagenase is usually needed to digest the extracellular matrix when creating a cell suspension from one tissue.
4. Excessive growth: When cells reach confluence, there is an excessive accumulation of cellular debris and a nucleic acid free of cell degradation.
When you are seeding your cells, make sure you pipette your cells in and out to detach any clumped cells, which are caused by trypsinization. Then, during the first 30mins of cell growth, do not move your plates as the cells are now trying to attach to the coverslips. Any slight movement will cause the cells to slide sideways and hence potentially clumping the cells.
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