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Adapting a Drosophila cell culture to Zebrafish for subsequent MACS-sorting - (Apr/24/2014 )

As the title states, I need to modify a protocol for cell-sorting Drosophila border-cells to be used for sorting out a desired cell-type in Zebrafish.

I have never worked with Drosophila nor done cell culture work before, so I would appreciate all the feedback.

 

1) Instead of "insect cell culture media" (Grace's media) used, I am guessing it would be advisable to use a mammalian cell culture media. So I am gonna go with DMEM.

 

2) I need to block the cells with some kind of serum. Because I want to later on stain the cells with antibodies that are conjugated to magnetic beads (the magnetic beads will be utilised for the magnetic bead-sorting when I sort out the desired cell type).

I have previously worked with regular blocking the tissues with goat-serum, then stained with antibodies and finally imaged with laser confocal.

But does the choice of using either goat-serum or bovine serum (BS) or fetal bovine serum (FBS) affect the procedure?

 

So I can either use

2a) first DMEM culturing and then block with goat serum

2b) DMEM-cultureing that is already containing BS or FBS

 

Which one is better? Is there any difference?

 

3)

The Drosphila protocol used both elastase and a cell dissociation buffer.

I know that you use elastase (or accutase or trypsin or any other protease-enzymes) as a way for breaking down the ECM and loosening the cells from each other as well as from the surface.

But what is the purpose of a cell dissociation buffer? Is it just a buffer that makes the proteases to work better?

Do you guys use such or do you just treat with a protease?

 

4)

Lastly I wonder about the compatibility of my antibodies and tissue. I haft to stain zebrafish tissues for the desired cell type. But the antibodies are generated in mouse (and the manufacturer does not provide any other species than mouse, rat and higher primate).

I know that when doing antibody-staining, you need to chose an antibody that has been generated in a species that is phyologentically as far apart/different from your species/tissue. For instance, use mouse-antibodies to stain rat/mouse-tissues.

But the gap between mouse and zebrafish is huge!! Would that work or just be completely incompatible to stain for?

-Biologystudent-

1) DMEM may work, a quick google search will probably tell you best.

 

2)Ideally blocking is done with serum from a different species to the antibody you are using.  FBS should work fine for this. Either A or B should be OK.

 

3)Dissociation is often done with trypsin or collagenase.  These should work OK in serum free medium.  Cell dissociation buffer should be OK too - this should maintain the cells in a reasonably healthy state.

 

4)This depends entirely on the antibody - some antibodies will work well in many different species, but others will not work.  Part of this is due to the protein sequence (epitope) which the antibody recognizes, conserved epitopes will be more likely to be recognized.  The only way to find out is to try it.

-bob1-

Thanks!

 

3)

What if I would go with a medium containing serum (instead of culturing with a medium that does not contain serum, followed by serum-blocking the tissues in a separate subsequent step).

Would the serum inhibit the action of trypsin? And if so, then would I need to have a culture dissociation buffer so that the trypsin still works despite the presence of serum?

 

1) DMEM may work, a quick google search will probably tell you best.

 

2)Ideally blocking is done with serum from a different species to the antibody you are using.  FBS should work fine for this. Either A or B should be OK.

 

3)Dissociation is often done with trypsin or collagenase.  These should work OK in serum free medium.  Cell dissociation buffer should be OK too - this should maintain the cells in a reasonably healthy state.

 

4)This depends entirely on the antibody - some antibodies will work well in many different species, but others will not work.  Part of this is due to the protein sequence (epitope) which the antibody recognizes, conserved epitopes will be more likely to be recognized.  The only way to find out is to try it.

-Biologystudent-

Serum will block the action of tryspin, but I don't know about collagenase. I don't think there will be too much collagen in serum, but there may be inhibitors present.  Serum can be added once the cells are dissociated from the extra-cellular matrix for the blocking step.

-bob1-