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We have used both 10K and 3K successfully (the lysine-fixable
dextrans), but the 3K is preferable.
On Jun 29, 2011, at 11:46 AM, JOEL B. SHEFFIELD wrote:
> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> 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:
>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> >
>>> *****
>>>
>>> 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
>>> www.invitrogen.com/**technicalsupport<http://www.invitrogen.com/technicalsupport
>>> >
>>>
>>>
>>>
>>>
>>>
>>> -----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
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> >
>>> *****
>>>
>>> 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:
>>>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> >
>>>> *****
>>>>
>>>> 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]
>>>
>>>
>>> Classification: UNCLASSIFIED
>>> Caveats: None
>>>
>>
>>
>> Classification: UNCLASSIFIEDCaveats: None
>>
>
>
>
> --
>
>
> 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
Gudrun Ihrke, Ph.D.
Research Assistant Professor
Department of Pharmacology (C2025)
Uniformed Services University School of Medicine
4301 Jones Bridge Road
Bethesda, MD 20814
phone office: (301) 295 3225
phone lab: (301) 295 3823
FAX: (301) 295 3220
email: [log in to unmask]
Classification: UNCLASSIFIED
Caveats: None
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