Fura 2 loading - Fura 2 /AM solution in DMSO loading in Tcells (Jan/16/2002 )
I am loading rat spleen lymphoctes (2 million) in a total volume of 1ml with 2micromolar Fura 2 concentration.The basal reading without Fura 2 comes around 60 at 340nm excitation and in loaded cells around 690.On addition of EGTA it decreases to aound 450.The cells do not show any increase in intensity at 340nm or any blew shift upon stimulation.I am not able to be sure whether cells are loading or not or whether the flourecence is totally external.If anyboby is using Fura 2 for loading cells please send me the deatailed protocol.
I would suggest using Pluronic Acid which can be bought from Molecular Probes. Usually mix 1:1 ratio of Fura-2 with Pluronic acid and load as usual. this should help the Fura-2 get into the cells. good luck!