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Hi,
Gudrun, by "small", what mw dextran are you suggesting?
Joel
On Wed, Jun 29, 2011 at 11:21 AM, Gudrun Ihrke <[log in to unmask]> wrote:
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> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> 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:
>
> *****
>> To join, leave or search the confocal microscopy listserv, go to:
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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
>>
>> ** vendor association acknowledged **
>>
>> Jason A. Kilgore
>> Technical Application Scientist
>> Molecular Probes Labeling and Detection Technologies
>> Cells Systems Division
>>
>> T 1 800 955 6288 then option 5 or 541 335 0353 . F 541 335 0238
>> 29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
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>>
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:CONFOCALMICROSCOPY@**LISTS.UMN.EDU<[log in to unmask]>]
>> On Behalf Of Gudrun Ihrke
>> Sent: Tuesday, June 28, 2011 12:26 PM
>> To: [log in to unmask]**EDU <[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:
>>
>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
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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
>>> --
>>> _ ____ __ ____
>>> / \ / / \ / \ \ Tobias I. Baskin
>>> / / / / \ \ \ Biology Department
>>> /_ / __ /__ \ \ \__ 611 N. Pleasant St.
>>> / / / \ \ \ University of
>>> Massachusetts
>>> / / / \ \ \ Amherst, MA, 01003
>>> / / ___ / \ \__/ \ ____
>>> www.bio.umass.edu/biology/**baskin<http://www.bio.umass.edu/biology/baskin>
>>> Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243
>>>
>>
>>
>>
>>
>> email: [log in to unmask]
>>
>>
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>
>
> Classification: UNCLASSIFIEDCaveats: None
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--
Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [log in to unmask]
URL: http://astro.temple.edu/~jbs
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