I want to observe my cells protein expression in calcium free condition. I am culturing the cells in normal DMEM media. Does anybody have the experience that show how I can make that media ca free with a detailed protocol?
You can buy calcium-free DMEM here:
https://www.lifetechnologies.com/order/catalog/product/21068028
Here is the complete formulation if you would like to make your own:
http://www.lifetechnologies.com/us/en/home/technical-resources/media-formulation.44.html
Alternatively, you can add a calcium chelator like BAPTA to your medium to sequester all of the free calcium.
http://www.sigmaaldrich.com/catalog/product/aldrich/a4926?lang=en®ion=US
EGTA is another option for an ion chelator, but it has some selectivity for magnesium as well.
Because your media Dulbecco's Modified Eagle's Medium (DMEM) already contains 1.8 mM calcium which is required for cell signaling, facilitates the attachment of cells to substrates and to one another and mediates many cellular events that affect cell movement, shape and three-dimensional structure and also in part of apoptosis. However, if you remove calcium form your cell culture media; you can treat media by: 1. Calcium Phosphates: Phosphorus is a prevalent element in cell culture systems that may contribute to calcium precipitation under some conditions. Inorganic phosphorus exists primarily as monobasic and dibasic phosphate at physiological pH. The approximate solubilities of calcium phosphates in cold water are monobasic phosphate, Ca(H2PO4)2, 71mM; calcium dibasic phosphate, CaHPO4, 1.7 mM and calcium tribasic phosphate, Ca3(PO4)2, 0.06mM. At pH 7.0, approximately 61% of the phosphate in the dibasic form and at pH 7.6, this increases to about 87%. The solubility of calcium phosphate in this form is approximately 1.7 mM and the highest concentration typically used in media formulations is 1.8 mM. This suggests that the precipitation of calcium by phosphate ions should only be a problem when the pH is made basic during activities such as base titration. The resulting formation of calcium tribasic phosphate would result in precipitation and possible loss of the calcium during filtration.
2. Chelators: Chelators such as citrate and EDTA are sometimes used in cell culture. EDTA has a log affinity for calcium of approximately 10.6 and it is often used to remove calcium from cell cultures media, especially when cells are being detached from substrates or when cell clumping is a problem. Citrate and albumin bind calcium with log affinities of approximately, 3.6 and 2, respectively. Citrate also binds to albumin. Together citrate and albumin may bind a considerable amount of the calcium in cell culture systems. Now it depends on your experimental workflow.
It"'s better to buy DMEM Ca free medium, because for addition of EDTA you should know the exact amount of EDTA required, otherwise it can have harmful effect on proliferation of cells. Even extra albumin can change the morphology of the cell.
If you add EGTA, be sure to adjust the pH because EGTA releases protons when Ca binds.
I am trying to make a calcium free "wash" and then add a solution with approx 1.5 millimoles calcium to cells before using Flexstation machine. Can anyone help with making these solutions? the volume in each of the wells of my 96 well plate has to be 200 microlitres.
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