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)


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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