Cell culture plates are treated so the plate surface is coated with groups negatively charged, and proteins of the cell surface can interact with them and attach very tightly. Thus, even when a cell is dying it remains attached to the surface (specially if the dead is fast), the interactions are very strong, that's why you can see dead cells adhered. Even, the fragments of a dead cells can ramain attached. Moreover, you are measuring dead cells with calcein and PI, and these molecules enter in the cell in early apoptosis, so you can see them stained, but they are dying, not dead, and so, they can remain attached.

In the other hand, cells need adhesion to survive long time, but cells can live without adhesion for some hours. Maybe you have detached some cells pippeting and they don't be necesarily dead or dying.

Thanks Pedro. I was not aware that the culture plates are treated for cell-adhesion. Your pointer was really helpful. Following is a link on Corning Life Sciences website which has references which explain it.

http://www.corning.com/lifesciences/us_canada/en/technical_resources/surfaces/culture/references.aspx

Ishan, under the right conditions some cells will even attach to teflon. While, it os true that TC polystyrene is 'treated' (i.e. treated with corona discharge or gas-plasma). These treatments generate oxygen radicals which oxidize and graft onto the polystyrene rendering the surface hydrophilic and negatively charged - once medium is added. John Ryan has written some excellent primers re cell culture. You can find these at www.sigmaaldrich.com/technical-documents/articles/biofiles/.

In reality individual cells do not 'see' surfaces. The net negative charge attracts proteins from the media (usually supplemented with serum) which act as 'adhesion factors'. That is molecules on the cell surface specifically recognise motifs within these adhesion factors and bind. Thus cell 'adhesion' is indirect.

For a review on the topic see: Tissue Eng. 2005 Jan-Feb;11(1-2):1-18.

http://www.ncbi.nlm.nih.gov/pubmed/15738657

in my experience with PC 12 cells, dead cells become round, detach from the bottom of the culture flaks and float in the medium

Another point to make: Trypan Blue is not a definitive assay for cell viability. It is subject to pinocytosis and phagocytosis and thus false negatives (alive but blue).

On the other hand, as long as you are aware of this, it is a convenient, quick and dirty test. The true test of cell viability is proliferation, although metabolic assays such as MTT, Alamar blue, etc, are useful. Please recognise, however, that ALL assays have limitations, and your interpretation must accommodate such individual limitations. Are you seeing what you think you are measuring?

Merely because a cell is floating, do not assume it is dead. It may have been detached by shear; it may be undergoing mitosis; it may have differentiated; it may be your media is cation deficient (e.g. RPMI); it may prefer to live in suspension!

And remember there are roughly 240 different cell types in the average mammal. Not all have identical needs, life cycles, or phenotype.

Thank you David and Dominique. I am a Mechanical Engineer by training, and learning more about biology to implement it into my research. I appreciate your pointers. I will post questions time to time to get information on what to read in order explain my observations.

I investigated the adhesion of granulocytes to a glass surface. The adhesion process can be described by a law similar to the Langmuir adsorption of dead particles to a substrate. The equilibrium adsorption coefficient can be determined by means of this law. Two processes are observed (Non-stimulated and stimulated cells). The equilibrium adsorption coefficient is a function of the concentration of migration stimulating molecules of the extracellular space. The percentage of loaded membrane-bound receptors is the essential cellular signal for the active adhesion process. In addition I found the process is cooperative.

We investigated the adhesion of human granulocytes to a glass surface (Z. Naturforsch. 46c 769 (1991)). The cell adhesion process can be described in analogy to the adhesion of molecules (dead material) to a substrate. The equilibrium adhesion coefficient can be obtained from this experiment. Two processes can be found (i) adhesion of non-stimulated cells and (ii) of stimulated cells. The adhesion is altered by migration stimulated molecules in the extracellular space. The essential signal for the stimulated process is the percentage of loaded membrane-bound receptors. The stimulated process is a cooperative phenomenon.

I gave a wrong reference in my previous answer. Here the correct one (Z. Naturforsch. 43C, 769 (1988)

Vascular endothelial cells in culture have a much higher rate of apoptosis and hence turnover than is generally appreciated - we looked at the effect of troglitazone (see publications) on both attached and unattached cell numbers over time - in that project we took the supernatant containing UNattached endothelial cells and when we put that media on new plates then some cells adhered and proliferated ... the point is that there is a lot to learn about cell adhesion and proliferation and apoptosis but with great respect to all the need is to do good science and not do cell culture research (i.e. to have a question).