PBS with or w/o Ca & Mg ??
Posted 05 May 2010 - 10:30 PM
Posted 08 May 2010 - 11:04 AM
if you have problems in washing tissue slices or fixed cells on glass slides you may use PBS with Ca2+ to force attachment
Posted 11 May 2010 - 07:21 AM
Posted 11 May 2010 - 02:34 PM
When to use Mg++ and Ca++, and when not to use
Adherent cells produce integrins (proteins) that aid attachment to the plate and each other. Mg & Ca are divalent cations that are essential to protein activity. If you want cells to adhere & grow, you want the cations. If you want to suspend them, you want to avoid/reduce the cations.
Trypsin /EDTA is used to loosen cells from substrate so they can be resuspended. Trypsin is a protease that digests the adhesion proteins that hold the cells to the plate. EDTA is a chelator that mops up divalent cations that are cofactors for protein activity. Calcium, especially, is essential to the proteins of cell signaling. By removing magnesium and calcium, cells lose contact with each other, there is no adhesion and they “round up” in solution.
We grow adherent cells in EMEM + FBS (for proteins, cations, essential growth factors). FBS contains proteins that naturally neutralize the trypsin. EMEM contains Mg & Ca.
When we want to detach the cells, we first rinse with Hank’s Balanced Salt Solution (HBSS) to wash cells prior to adding trypsin because it does not contain magnesium or calcium and it will reduce the concentration of those cations as well as the FBS proteins.
After the cells are re-suspended, we draw out a volume of cells and put them in a flask with the EMEM + FBS growth media. It is IMPORTANT to neutralize the Trypsin ( we do this with the media,) or risk digestion of intercellular proteins & cell death.
Edited by Whitac, 11 May 2010 - 02:35 PM.