Hi all, I am using the Super Ecliptic pHluorin-GluA1 (or 2) probe to study the dynamic of AMPAR. Yet the treatment I am using as a positive control (chemical LTP: glycine, bicuculine, strychnine) seems to quench the fluorescence instead of increase it. Do you have any experience or knowledge about what can induce a change in surface AMPAR (even when you used other assays)? I am looking for a pharmacological treatments that can help me confirm whether I can biologically chance the level of SEP-GluA signal. For now, I am using amonium chloride and MES, one is to turn on everything and the over is to quench surface signal, but they are not very biological...
Thank you all!
Huong
Hi Huong Ha. A quick guess. If you get very strong post-synaptic depolarization and calcium entry then the cytoplasmic pH decrease (due to proton-Ca++ exchange on CaBP) is probably masking the comparatively much smaller increase of surface fluorescence (exocytosis). You should have a look at Ric Huganir's paper in PNAS on this topic, for in-depth demonstration of this. Obviously you cannot reduce your [Ca]o but increasing membrane-permeable pH buffer (bicarbonates) may help. Good luck.
Hi Thomas,
That is also my guess. I saw several papers about pH decrease induced by NMDAR activation. I was thinking of increasing the pH buffer as well. Yet at the moment my bath solution is with HEPES, not with bicarbonate since we are imaging on culture neurons and do not bubble the solution. :-? Thank you for the suggestion! I will look more into Huganir's work.
Huong
Dear Huong,
You could add bicarbonate together with HEPES, without bubbling. Let it equilibrate at 37deg under atmospheric pCO2 (0.04% and rising :-( ), before final adjustment. In the Krebs-Ringer the usual concenration is 20mM HEPES for 5mM Bicarb. I hope this helps.
Dear Huong, I see that you voted my answer up. Did it work for you ? A colleague just mentionned membrane-permeable HEPES or MOPS as a stronger alternative (AM-HEPES) but I haven't found a supplier for this. The synthesis is supposed to be easy... Kepp us posted. Cheers.
Hi Thomas, I actually have not had a chance to try your method. I moved to a new lab last month and then had a fairly long winter break. Will keep you posted for sure! And I will keep an eye out for the membrane permeable buffers that you just mentioned. Sounds very useful!
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