beads for localized stimulation of proteins

[abhijit debroy]

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Hi,

This isn't a confocal problem, but I have seen great suggestions for many
imaging issues so I figured I'll post mine.

I am trying to image the effects of a localized ligand stimulation on
migrating cells (osteoblasts). To localize the ligand, I am using protein
A/G coated magnetic beads (1um dia). However the beads are aggregating, and
moving around too much when imaging. Also they are completely messing up
the DIC image (not too important apart from knowing the location of the
bead and the region of contact.)

I was wondering if any of you have any suggestions to make this set up
work. Ideally, the beads should be ~2-5 um diameter, have either some
functional group which I can use to immobilize the protein and be heavy
enough that it settles down in a culture and doesn't move about too much.
Also, something that doesn't interfere too much with fluorescent imaging
(438 nm and 515 nm currently) would be great.

I apologize for the rather long post.

Thanks
Abhijit Deb Roy
PhD Candidate
Biomedical Sciences Program
University of Connecticut Health Centre, Farmington

[Aleem]

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Abhijith,

Have you tried localizing beads with poly lysine. I do lot of fluorescence
imaging of beads, poly lysine coated coverslips worked well for me. To
avoid the aggregation try sonication or votexing.

Hope it helps,

Aleem



On Tue, Jun 4, 2013 at 11:11 AM, abhijit debroy <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> This isn't a confocal problem, but I have seen great suggestions for many
> imaging issues so I figured I'll post mine.
>
> I am trying to image the effects of a localized ligand stimulation on
> migrating cells (osteoblasts). To localize the ligand, I am using protein
> A/G coated magnetic beads (1um dia). However the beads are aggregating, and
> moving around too much when imaging. Also they are completely messing up
> the DIC image (not too important apart from knowing the location of the
> bead and the region of contact.)
>
> I was wondering if any of you have any suggestions to make this set up
> work. Ideally, the beads should be ~2-5 um diameter, have either some
> functional group which I can use to immobilize the protein and be heavy
> enough that it settles down in a culture and doesn't move about too much.
> Also, something that doesn't interfere too much with fluorescent imaging
> (438 nm and 515 nm currently) would be great.
>
> I apologize for the rather long post.
>
> Thanks
> Abhijit Deb Roy
> PhD Candidate
> Biomedical Sciences Program
> University of Connecticut Health Centre, Farmington
>

--
*Best Regards,
*
*Aleem Syed
*
*Graduate Assistant
*
*Emily Smith Research Group
*
*0712 Gillman Hall
*
*Department of Chemistry
*
*Iowa State University
*
*Ames Laboratory
*
*IA, USA
*
*Work Ph. # 515-294-8586
*
*Cell Ph. # 515-708-8283
  *

[abhijit debroy]

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I coat my coverslips with fibronectin as the cells appear to like that. I am concerned that vortexing or sonicating the beads with the ligand may shear the ligand.

What kind of beads do you use? Do they remain relatively steady in culture conditions?

Thanks
Abhijit

On 4 Jun 2013, at 13:21, Aleem Syed <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Abhijith,
>
> Have you tried localizing beads with poly lysine. I do lot of fluorescence
> imaging of beads, poly lysine coated coverslips worked well for me. To
> avoid the aggregation try sonication or votexing.
>
> Hope it helps,
>
> Aleem
>
>
>
> On Tue, Jun 4, 2013 at 11:11 AM, abhijit debroy <[hidden email]>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi,
>>
>> This isn't a confocal problem, but I have seen great suggestions for many
>> imaging issues so I figured I'll post mine.
>>
>> I am trying to image the effects of a localized ligand stimulation on
>> migrating cells (osteoblasts). To localize the ligand, I am using protein
>> A/G coated magnetic beads (1um dia). However the beads are aggregating, and
>> moving around too much when imaging. Also they are completely messing up
>> the DIC image (not too important apart from knowing the location of the
>> bead and the region of contact.)
>>
>> I was wondering if any of you have any suggestions to make this set up
>> work. Ideally, the beads should be ~2-5 um diameter, have either some
>> functional group which I can use to immobilize the protein and be heavy
>> enough that it settles down in a culture and doesn't move about too much.
>> Also, something that doesn't interfere too much with fluorescent imaging
>> (438 nm and 515 nm currently) would be great.
>>
>> I apologize for the rather long post.
>>
>> Thanks
>> Abhijit Deb Roy
>> PhD Candidate
>> Biomedical Sciences Program
>> University of Connecticut Health Centre, Farmington
>
>
>
> --
> *Best Regards,
> *
> *Aleem Syed
> *
> *Graduate Assistant
> *
> *Emily Smith Research Group
> *
> *0712 Gillman Hall
> *
> *Department of Chemistry
> *
> *Iowa State University
> *
> *Ames Laboratory
> *
> *IA, USA
> *
> *Work Ph. # 515-294-8586
> *
> *Cell Ph. # 515-708-8283
>  *

[Aleem]

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I am afraid, I don't use any culture media on top of the beads. I use
polymer beads as standards for fluorescence imaging. I do sonicate ligand
coated quantum dots all the time and  never saw any ligand dissociation,
Though you need to optimise the temperature for sonicatoin and
concentration for your application, generally lower temperatures (ca. 10-18
degree Celsius) work fine.

Best,
Aleem

[abhijit debroy]

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Thanks Aleem. I'll try sonicating the beads for dirpersion.

Cheers
Abhijit


On Wed, Jun 5, 2013 at 10:20 AM, Aleem Syed <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I am afraid, I don't use any culture media on top of the beads. I use
> polymer beads as standards for fluorescence imaging. I do sonicate ligand
> coated quantum dots all the time and  never saw any ligand dissociation,
> Though you need to optimise the temperature for sonicatoin and
> concentration for your application, generally lower temperatures (ca. 10-18
> degree Celsius) work fine.
>
> Best,
> Aleem
>

--
Abhijit Deb Roy
Biomedical Sciences Program
University of Connecticut Health Centre, Farminton