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I second that advice, though I imagine the timing would depend upon the cell line and environmental conditions.
Gudrun, what was the cell line and media you used?
Jason
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Gudrun Ihrke
Sent: Tuesday, June 28, 2011 12:26 PM
To: [log in to unmask]
Subject: Re: endocytosis for cell biology lab
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Tobias,
Unless you preincubate with lipoprotein-deficient serum, the LDL
receptor will be downregulated. It would be much easier to use a small
fluorescent dextran to label the endocytic pathway towards lysosomes:
~3 min incubation will give you early endosomal staining, ~15-45 min
late endosomal (best use a pulse of 10-15 min pulse and chase 15-30
min for bright staining, >1h late endosomal/lysosomal staining (use
also a pulse if you want to chase the dextran out of earlier
compartments).
Gudrun
Gudrun Ihrke, Ph.D.
Research Assistant Professor
Department of Pharmacology (C2025)
Uniformed Services University School of Medicine
4301 Jones Bridge Road
Bethesda, MD 20814
On Jun 28, 2011, at 2:25 PM, Tobias Baskin wrote:
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>
> Hi all,
> This is not a "confocal" question but rather a cell bio/microscopy
> related one. Nevertheless, I am hoping someone will be able to answer.
>
> I co-teach a cell biology lab course where students learn how to
> use conventional epi fluorescence and video microscopy. We have a
> variety of "cells in action" labs for them to do, one of these being
> endocytosis. As markers, e have used labeled-transferrin and also
> DiI labeled LDL, with the idea being that the transferrin is
> recycled back to the plasma membrane whereas the LDL moves into
> lysosomes. While the transferrin labeling seems to work more or less
> as expected, results with the DiI LDL are erratic. Sometimes there
> is no labeling at all, and we never have seen what looks like deep
> internal labeling in putative lysosomes.
>
> We plan to troubleshoot later this summer but as none of us have
> studied endocytosis, we have no first hand experience. I am
> wondering if anyone out there knows of a tried and true endocytosis
> cell bio lab? Or perhaps knows about some tricks for handling DiI
> LDL. Or perhaps it is a matter of a good cell line -- we work
> typically with a 3T3 fibroblasts or LLCPK epithelial cells (with
> equally dismal results for the DiI LDL endocytosis).
>
> Thanks in advance for any suggestions.
>
> As ever
> Tobias Baskin
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