When I wash cells with PBS, what happens? Why does PBS sometimes cause confluent cells to peel off? Is washing with PBS always necessary before splitting or freezing cells? What is the functional difference between PBS with calcium and magnesium and PBS without those minerals?
Before splitting and freezing cells grown in a monolayer, you need them to detach from the culture surface. For this you are most likely to use trypsin. Before you add it to the cells you need to wash away the cell culture media, since trypsin would also break down proteins that are present in the media, like FCS, reducing its activity before it reaches the cells. The reason why we use PBS and not, let's say, water, is that its osmolarity and pH make it a physiological buffer that will not harm the cells.
It is not the PBS that causes the confluent cells to peel off, any other liquid would do the same if the cells are only weakly attached. To get a single cell solution, which is necessary for both splitting and freezing, you will probably always need the PBS wash step, sometimes twice. If losing the cells in this wash step is a real problem, then simply collect the PBS, and try to dissociate the cells by pipeting up and down. You can check how successful this was when you count the cells and don't see any clumps.
Dear Sir
I think the links below will benefit you in your search
http://www.rapidvet.com/pdf/cellwashproc.pdf
https://www.iitk.ac.in/biomaterialslab/Bacteria%20Culture_related_Info.pdf
https://books.google.com.ly/books?id=TPNzgOqnYcMC&pg=PA82&lpg=PA82&dq=PBS+clean+cells&source=bl&ots=MDm-xNxGyR&sig=x0YL4nLulc1_AQIJsmMsPjtwH_c&hl=ar&sa=X&ved=0ahUKEwjpwrqd9ZTcAhWFAJoKHW3GDTIQ6AEITDAG
Best Regards
Your questions may be answered by Amer Basile under this ResearchGate question: https://www.researchgate.net/post/Why_are_calcium_and_magnesium_free_PBS
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