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Cell-cell interaction using facs aria

Cell-cell interaction using facs aria - Flow Cytometry Forum

Cell-cell interaction using facs aria - Flow Cytometry Forum. Discuss FACS cell sorting, flow cytometry, analysis and cell staining.


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Old 05-01-2010, 01:08 AM
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Default Cell-cell interaction using facs aria



Hi all,
I am phd student, my experiment involves binding of epithelial cells to yeast. I can see this clearly through microscope when i mix the cells together and incubate them.

so now i am trying to quantify this binding by using the facs aria, and i am a complete newbie to flow cyto. Additionally i do not want to label these cells with differenet flourescent dyes. I am just trying to use autofluorescence and scattering properties of the cells.

I am tried fsc vs ssc plots for just the yeast (~7um) and the epithelial cell (~80um) but unfortunately although there is such a huge difference in size, there scattering plots overlap, so does they autofluoreescence.

I am wondering if i could change parameters to change the scattering properties.

Would be happy to hear other suggestions as well.

cheers
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Old 05-04-2010, 03:27 PM
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Default Re: Cell-cell interaction using facs aria

Honestly I don't think you'll be able to get good results without labeling the cells in some way. FSC/SSC should be able to distinguish the different sizes, but cell debris in your mammalian cells will look like yeast, and clumps of yeast cells will look like mammalian cells, so I think you're always going to have a huge amount of error if you're just looking at FSC/SSC.

I don't know jack about yeast, but I assume there exists some cell-permeable nuclear dye or simple surface dye that you could use to quickly stain live yeast. If you then stain your epithelial cells with a different dye (DAPI, acridine orange, whatever), you can look for colocalization of the two dyes to determine cell binding. The dyes are cheap and this kind of staining takes like 10 minutes, so I can't see any reason not to do it.....

A second problem, though, is that FACS generates pretty large shear forces on the cells, so if the binding is weak, your cells may detach from each other while going through the machine. You may need to fix and crosslink them in some way before running them though.
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Old 05-05-2010, 07:09 AM
Pipette Filler
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Default Re: Cell-cell interaction using facs aria

Quote:
Originally Posted by Bonnie View Post
Honestly I don't think you'll be able to get good results without labeling the cells in some way. FSC/SSC should be able to distinguish the different sizes, but cell debris in your mammalian cells will look like yeast, and clumps of yeast cells will look like mammalian cells, so I think you're always going to have a huge amount of error if you're just looking at FSC/SSC.

I don't know jack about yeast, but I assume there exists some cell-permeable nuclear dye or simple surface dye that you could use to quickly stain live yeast. If you then stain your epithelial cells with a different dye (DAPI, acridine orange, whatever), you can look for colocalization of the two dyes to determine cell binding. The dyes are cheap and this kind of staining takes like 10 minutes, so I can't see any reason not to do it.....

A second problem, though, is that FACS generates pretty large shear forces on the cells, so if the binding is weak, your cells may detach from each other while going through the machine. You may need to fix and crosslink them in some way before running them though.
Thanks for your response Bonnie..

I have tried the dye approach before to visualise the binding of the yeast and Buccal cells through a confocal microscope. The dyes I tried were DAPI/SyBr green/Hoescht to stain yeast and fm64 to stain Buccal cells. What I noticed was that even after considerable amount of washing when the cells are interacting, they seem to pick up the other dyes, so I can see the yeast staing red with the fm 64 and the nuclic acid dye ( DAPI/SyBr green/Hoescht) and the Buccal cells also picking the nuclic acid dye. So I avoided this approach as there would be cross talk. Alternatively I could use covalent linking dyes but I am worried about the non specific binding/ inhibition of the interaction.

Any suggestions would be appreciated.

cheers
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