Use of fura-2/AM to measure intracellular free calcium in Selenomonas ruminantium

Abstract

This paper describes a procedure for loading the acetoxymethyl ester of fura-2 (fura-2/AM), and the subsequent measurement of the concentration of intracellular free Ca2+ ([Ca2+]1) in Selenomonas ruminantium (S. ruminantium,) using this technique. To ascertain the optimal loading conditions, the effect was examined on the loading of fura-2/AM of ethylenediaminetetraacetic acid (EDTA), lysozyme, pluronic F127 alone, or the simultaneous application of EDTA and pluronic F127. Individual administration of either EDTA, lysozyme or pluronic F127 did not produce an optimal loading of fura-2/ AM. The co-application of pluronic F127 and bovine serum, albumin after treatment with EDTA increased the ratio of the fluorescence due to excitation at 340 nm to that at 380 nm ( R340/380) markedly, with a high signal intensity for intracellular fura-2, indicating that adequate loading had been obtained. Using the present loading method, it was found that the resting free [Ca2+]I of S. ruminantium was 48.1±2.2nM (n = 6). This is approximately one half that found in Eschrichia coli, Propionibacterium acnes, Streptococcus bovis and eucaryote cells. This is the first measurement of free [Ca2+]1 using fura-2/AM in the Gram-negative strict anaerobe S. ruminantium.