XB-IMG-120627
Xenbase Image ID: 120627
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Figure 5. Measurement of pHi in Cx46 expressing Xenopus oocytes during application of medium (A) equilibrated with 100% CO2 and (B) adjusted to pH 6.3 with HCl. pHi was monitored with BCECF and recorded at five distances from the edge of the oocyte, ranging from 50 to 450 μm. Experimentally, these values were obtained by placing 100-μm circular “regions of interest” (ROI) centered at the specified distances on the acquired image of the oocyte and replaying the saved acquisition. During image acquisition, the objective was focused on the oocyte's perimeter. With a large, round oocyte, this focal plane would pass through the center of the oocyte, although with considerable light scatter. The ratiometric determination of pH in this plane will include some signal from the periphery above and below the center of the oocyte, but the image from the perimeter of the oocyte will include entirely peripheral cytoplasm. Thus, there is a significant difference in the amount of signal contributed from deep within the oocyte cytoplasm as the ROI is moved away from the perimeter. The slower change in pH towards the center of the oocyte was observed consistently, but eventually the pH reached the same value in the center as at the periphery. (A) A 4-min application of 100% CO2-equilibrated medium lowered pHi from ∼7.6 to ∼5.8. An enlarged view (A1) shows how the kinetics of intracellular acidification slows with increasing distance from the oocyte periphery. The same steady state value of pHi was reached in all cases before recovery was initiated by washing. Similar kinetics was seen with recovery (A2). (B) An ∼3-min application of HCl-acidified pH 6.3 medium also lowered pHi. Again, the effects were faster at the edge of the oocyte. With the duration of the application shown, pHi decreased less at the center of the oocyte. Image published in: Trexler EB et al. (1999) Image reproduced on Xenbase with permission of the publisher and the copyright holder. Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View |