Stem cell side population detection

Hi Flow-ers,

 

I would like to ask for your advice on the determination of stem cell
side population in cultured cell line.	I have adapted the Godell's
protocol for staining but using a similar dye, DyeCycle Violet from
Invitrogen so that the stained cells can be excited by a violet laser.
The instrument I am using is an FACS Aria which does not have an UV
laser.

 

1.	In the attached power point, the first slide showed the dot plot
of DyeCycle Violet (blue vs. red).  The cells were treated +/-
Verapamil.  It seems to me that the dim population disappeared with the
addition of verapamil, suggesting it may be the side population (SP),
indication of stem cell like.  However, the scale I used to detect the
dim population was on log, rather than linear as most literature
suggested when using Hoechst 33342 and LSR cytometer.  I tried using
linear scale to look at the fluorescence but the cells were scattered
all over.  I am concerned that I did not see the typical "tail" of the
SP in most literature.	Could anyone advise if the difference is due to
the dye and instrument ?  Or am I really looking at the side population
? 
2.	The second slide contains the FSC and SSC data of the same
samples listed in 1)  The cell population I am looking at came from
Medullablastoma, DAOY which is fairly big (average diameter ~ 20u).  I
have to lower voltage of FSC to 100 and SSC to 200 in order to put the
cell bodies in frame.  Could anyone also advise if the voltage I am
using is acceptable ?  I am concerned the low voltage may compromise the
resolution of my cell population. 

 

Thanks very much in advance for your help as always.

 

Regards,

 

Albert 

Exelixis Inc.

 

 



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