Can someone advise me on the correct preparation of ACSF for organotypic slicing?

08 January 2015 0 3K Report

I am preparing ACSF for organotypic slice culture experiments (which I haven't done before) but I want to make a 10x stock and I want to make sure I have separated the correct components as some protocols vary over this (i.e. leaving out bicarbonate or glucose from the stock). Having said that, I would feel more comfortable making up fresh 1x ACSF dailly.

Also, is it necessary to adjust the pH of ACSF before or after bubbling with carbogen? I would assume beforehand?

My stock solution is (mM): 125 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 17 glucose. Bubble in carbogen for 15-20 minutes. pH 7.3-7.4. Store at 4oC.

I then dilute to 1x and add CaCl2 and MgCl2 to a final conc. of 2 and 1mM respecitvely, then constantly bubble with carbogen until use.

Thanks in advance!

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