Re: endocytosis for cell biology lab

Posted by Gudrun Ihrke on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Concentrating-bacteria-cells-for-microscope-visualization-tp6519025p6529393.html

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Jason,
The times should hold up pretty well for many mammalian cell lines,  
provided the cells are incubated at 37C and have prominent clathrin-
mediated endocytosis. We have used fibroblasts or epithelial cells.  
Fluid phase uptake is of course through all endocytic pathways, but I  
have little experience with cells that use mostly other uptake  
mechanisms.  We have used either normal serum-containing tissue  
culture medium (buffered with HEPES if pH is a concern due to small  
volumes, and/or preequilibrate the dextran-containing  medium in CO2  
incubator) or BSA-containing medium for the pulse.

Regards,
Gudrun

On Jun 28, 2011, at 4:05 PM, Kilgore, Jason wrote:

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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:[hidden email]
> ] On Behalf Of Gudrun Ihrke
> Sent: Tuesday, June 28, 2011 12:26 PM
> To: [hidden email]
> 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
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>
> 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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