Dear List, I would like to pose a question regarding the use of 5(6)-carboxy fluorescein diacetate, succinimidyl ester (5(6)CFDA/SE). This question does not relate at all to the current CFSE discussion on the list. I was planning to use CFDA/SE to enumerate viable bacteria from water samples by flow cytometry, where viability is obviously based on the presence of active internal esterases that cleave the molecule into its fluorescent carboxy fluorescein derivative. I have made a stock solution of CFDA/SE (25mM) in high quality, fresh, anhydrous DMSO (stock solution stored in a desiccator). I have been trying to optimise the staining conditions by testing different buffers and varying pH (buffers include PB, PBS, MilliQ water and also seeing if the addition of EDTA facilitates permeabilisation of the cells for better staining). However, after the addition of CFDA/SE (both 50 and 100uM final concentration) to the bacterial suspensions at 35oC, I noticed that after about half an hour the solutions started to turn a bright yellow/green colour. I thought that this was due to the bacteria taking up the molecule and cleaving it internally, and as a result some of the fluorescent product was leaking out of the cells into the supernatant. I examined the suspensions by epifluorescent microscopy (blue light excitation) and I was nearly blinded by the intensity of the background (ie. the supernatant was fluorescent, tentatively indicating that the yellow/green colour of the suspensions was due to the cleaved fluorescent product of CFDA/SE). Having seen this, I set up a series of negative controls, ie. all the different buffers without the addition of the bacteria. I then added CFDA/SE at the same concentrations as before to the negative controls. To my surprise, after half an hour at 35oC incubation, the negative controls started to turn the yellow/green colour. To further investigate this, I set up the negative controls once again, this time in a real-time PCR machine set to detect fluorescein fluorescence. After the addition of CFDA/SE to the negative controls, the real-time PCR maching started to detect fluorescence in the phosphate buffer [pH 8.0] negative controls within 30 seconds. After 2 mins, all of the buffers were producing a fluorescent product and within 5 mins the PB solutions had saturated the fluorescent detector. The fluorescent intensity and rate of fluorescent product production was proportional to pH of the buffers where the buffers at a pH of 8.0 produced the fluorescent product faster than buffers at 7.5 and 7.0. Therefore, it seems clear to me that the addition of CFDA/SE to the aqueous buffers is causing the molecule to break down into its fluorescent derivative. The manufacturer states that if CFDA/SE is stored in the presence of even minimal water, it will break down over time (therefore CFDA/SE is obviously not stable in an aqueous medium). And even when stored appropriatley, CFDA/SE is said to only be good for 6 months but this isn't an issue with my work because I am using new CFDA/SE and storing it in anhydrous DMSO. Therefore, it appears to me that the molecule is breaking down upon the addition of it to an aqueous solution such as the buffers. If this is the case, obviously I can't use CFDA/SE for bacterial viability studies. I have read many papers that use CFDA/SE for bacterial viability studies and I am basically following the same protocol as they report to be successful. I was therefore wondering if anyone has seen this occur before or does anyone have any idea as to why this is happening or what infact is happening. Have I just got a 'dud' batch of CFDA/SE from the manufacturer? Any comments or suggestions would be appreciated. Daniel Hoefel
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