CELL MIGRATION QUESTIONS-CLARIFIED

bunny bunny at cotleur.com
Mon Oct 17 08:30:16 EST 2005


Just to clarify:
I would like to enumerate the migrated cells:
   1. without removing them from the well  (I want to eliminate the 
variability introduced by trying to recover each cell in each well).
   2. More accurately than simply counting fields in a microscope   
(reduce the variability due to subjectivity/human eye )
   3. via a method that can be scaled up for higher throughput (ie- 
examine migration patterns with more wells/conditions simultaneously)

Thanks!
bunny

Bunny Cotleur, M.S.
Sr Research Technologist, Dept of Neurosciences
Scientific Consultant, Flow Cytometry Core
Cleveland Clinic Foundation
Lerner Research Institute, NC30
9500 Euclid Avenue
Cleveland, OH 44195
Lab: (216)444-1164
*********************************************
Caminante, no hay camino.      Se hace camino al andar.




> I have a detection problem that is tangential to flow.....
>
> I am attempting to enumerate PBMC's migrating through an epithelial 
> cell layer in response to hormones (using the Transwell system) after 
> a 5hr incubation. I put a million cells in the top chamber and can 
> recover sufficient migrated cells to stain for phenotyping via flow 
> cytometry. (It's sloowwww flow, but it works!) Using flow I can 
> compare the input, migrated and nonmigrated populations to see which 
> cell types respond.
>
> The problem is: I would like to have more precise counts of the actual 
> NUMBER of cells that migrate. So I have been experimenting with both 
> calcein am and CFSE (separately!) as a way to label the cells, then 
> detect the migrated cells via microplate fluorimeter. (This way I 
> reduce the error from trying to recover every last migrated cell). SO 
> far I'm running parallel cultures- 1 for flow, one for cell counts. 
> {It would be nice if eventually I could also stain the cells for flow 
> after counting them in the fluorimeter-but not absolutely necessary}.
>
> I incubate the cells with either probe (as product insert directs), 
> wash several times, and then dispense the cells into 24well plates at 
> 10*7,10*6,10*5, 10*4, 10*3, and 10*2 cells per well. Then I read on a 
> fluorescent plate reader at the appropriate excit/emiss. On the 
> highest 2, I can see a nice emission (over background media alone).
>
> So far I have been really disappointed in the ability to see any 
> differences between the number of cells/well. Although I didn't expect 
> to be able to count 100 cells in this system, colleagues have claimed 
> to be able to enumerate the number of migrated cells in the thousands. 
> I see very little difference between 10*7 and 10*6, and then the 
> emission levels off. (I check the plates on a microscope after the 
> plate reader- and yes, there are plenty of cells!)..
>
> Any advice?
>
> Given I've tested CFSE to 5uM- I plan to try 10uM today. At what point 
> does CFSE become toxic? (My cells need to be happy for 5hr post 
> labelling).
>
> I have the cells suspended in PBS/BSA/Hepes (no phenol red) but for a 
> 5hr culture I was hoping to used my defined media (XVivo15, phenol-red 
> free). But is has enough of a yellow cast that I'm also worried it 
> would interfere with the detection. Is this a problem?
> The manufacturer of the reader (Spectramax) says that tissue culture 
> plates are ok in this system, but the empty wells seem to have as much 
> "background fluorescence" as media wells!
>
> Colleagues here have been using calcein am, but I read that it begins 
> to leak back out of the cells after 3hr. (I also could not detect any 
> labelled cells below 10*6 with calcein)
>
>
> Is anyone doing any similar experiments that they could share 
> advice/wisdom? Any small nugget would be GREATLY appreciated!
> Thanks-
> bunny




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