pH sensitive probe
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Hi,
I am looking at intracellular pH of yeast cell using a dye called SNARF-4F. In order to measure the internal pH I have created a calibration curve. This done by loading cells with SNARF-4F and then added 30 uM Amphotericin B (an ionophore) to equilibrate internal and external pH and then placing them in buffers (5-7.8) pH. However, from my initial results the amphotericin does not seem to equilibrate pH completely. Does anyone have a good method for killing yeast cells, without tearing the plasma membrane or cells wall (as the fluorophore would leak out)? I am not sure heating is a good idea as the fluorophore might sustain damage during heating. ------------------------------------------------------------- Thomas Aabo Phone: +45 35333636 M.Sc., Ph.D student, [hidden email] University of Copenhagen Department of Food Science Rolighedsvej 30 1958 Frederiksberg C Denmark www.ifv.life.ku.dk ------------------------------------------------------------- |
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Dear Thomas,
is fixation/permeabilization maybe an option, or do you think this will alter the measured intensities? Regards, Dries. 2009/6/19 Thomas Aabo <[hidden email]> Hi, -- Dries Vercauteren, PhD student Master of Bioscience Engineering: Cell and Gene Biotechnology Ghent Research Group on Nanomedicines www.ugent.be/fw/en/research/biofys Faculty of pharmaceutical sciences, Ghent University Harelbekestraat 72, 9000 Ghent Belgium Phone: +329/264 80 49 Mobile: +32485/30 69 80 E-mail: [hidden email] [hidden email] |
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In reply to this post by Thomas Aabo
Dear Thomas,
We used SNARF-1 with the ionophore nigericin in mammalian cells. So a little different to your work. We had to ensure that 20uM nigericin was in ALL the calibration buffers otherwise it seemed to wash out of the cells.
Also I eventually changed our techniques from changing solutions to simply bubbling with different CO2 / Air mixtures and measuring the pH of the solution directly with a pH meter. This gave much less variability in the calibration curves than changing solutions. Although I did not have one at the time, the "CO2 Gas/pH Controller" by Harvard Apparatus is ideal for this type of calibration.
It has a small pH electrode that can be placed in the buffer; by setting the desired pH set point, the instrument will adjust CO2 flow so that the desired pH is achieved.
The calibration buffer had Bicarbonate as well as a little bit of hepes to ensure the pH did not fluctuate wildly at low or zero CO2 concentrations.
No commercial affiliation with Harvard or Invitrogen.
All the best,
Steve
Stephen H. Cody
Imaging Research Fellow & Manager Monash Micro Imaging – AMREP Monash University 6 Floor Burnet Tower Alfred Medical Research & Education Precinct 89 Commercial Rd, Melbourne, Australia, 3004 www.microimaging.monash.org 2009/6/19 Thomas Aabo <[hidden email]>
Hi, |
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