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Lysosomal ions


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#1 Dwolfe38

Dwolfe38

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Posted 26 January 2011 - 11:57 AM

Hey all, I have no idea if this is the ideal forum subheading for this question, but since most of the lysosomal questions popped up here, I figured I'd give it a shot.

I have a two parter.

First, I want to measure lysosomal pH in live cell culture. I know that reagents like lysosensor and oregon green display pH dependent fluorescence and that the bulk of the papers using these determine exact values microscopically, so my first question is has anyone tried to quantify lysosomal pH using a fluorimeter instead? I find that my experiments result in a standard curve (salt solution + ionophores pHd accordingly) that tends to be far lower in its ratios than my samples, making exact quantification difficult and not exactly reproducible. If anyone has been able to successfully navigate this problem and could suggest a good method for rectifying this problem I'd appreciate it. The dextran conjugated forms of these reagents work much better, but are SIGNIFICANTLY more expensive than the unconjugated.

Secondly, Can anyone suggest a good reagent/protocol for determining lysosomal chloride levels? I've read some papers using SPQ, but I was wondering what other options might be available to me. I should note that I am not even remotely qualified to attempt patch-clamp at this point, so I'm hoping for a fluorimetric/colormetric approach.

Thanks a ton,

- D




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