We haven't ever done pH in microorganisms but have done so in human blood cells - platelets, granulocytes, now doing it in monocytes, and in phagocytic compartments (all are published). In all cases we used BCECF and calibrated using nigericin. However, that has to be done in a buffer system whose K+ concentration mimics that of the interior of the cell, because nigericin collapses the ratio of H+in/H+out to equal that of K+in/K+out, which therefore needs to equal one. That seems to be the thing people who've contacted me in the past don't take into consideration. If one takes that precaution, we have had very, very reproducible calibration curves. Elizabeth R. Simons joerg ueckert wrote: > > Folks, > > We're holding us busy by applying CFDASE for ratiometric determination of > internal pH in bacteria and yeast. Apart from strain- and pre-history - > dependent difficulties in dye uptake the generation of a 'useful' calibration > curve is the biggest challenge. The well-known combination > valinomycin/nigericin results in a high degree of variability and curve shifts > upon repetition. Also, apart from these, other agents (e.g. alcohols) or > methods (e.g. electroporation) to facilitate equilibration of internal and > external pH give stunning differences compared to mentioned 'standard' method. > All remarks, suggestions and experiences with agents which provide reliable > calibration curves for pHi in microorganisms are welcome. Providing a > sufficient number of responses, I will post a summary on the list. Thanks in > advance. > > - Joerg > > ------------------------------------------------ > Joerg Ueckert > Unilever Research > Microbiology > Vlaardingen, NL > > Email joerg.ueckert@unilever.com > ------------------------------------------------ -- Elizabeth R. Simons, Ph.D. Professor of Biochemistry Boston University School of Medicine 715 Albany Street, K 602 Boston, MA 02118 phone (617) 638-4332 FAX (617) 638-5339 email: esimons@bu.edu
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