Re: Stem cell side population detection

Hi Albert,

SP detection of putative stem cells is a bit of a hot topic still and
clearly as more people are using violet or near-UV lasers then things may
not look quite the way they do with the UV Hoechst excitation used in the
original papers.

I know that several groups have been using the DyeCycle dyes to look at SP
and Invitrogen may be able to provide some practical details. I would
question the machine set up; it is a good idea to be able to see the cell
cycle distribution as well as the SP and to do this you really should use a
linear amplification. Also it is very important - particularly in cell lines
- to do some experiments where you vary the dye concentration and length of
time of incubation (this is certainly true of Hoechst - I assume its the
same for the DyeCycle Violet). With experience you know when you are
reaching staining equilibrium and you can then be more convinced that a
visible SP that disappears with your blocking agent is due to the action of
membrane pumps.

With cell lines, we have also found that the degree of confluence will
affect the SP and, as you are probably aware, it is very important to keep
the cells happy at all stages of the preparation so the medium they are in
is also important and can influence the results. Again with cells lines we
don't always see the 'classical' SP-shape but removal of a defined
population by verapamil or reserpine can make a good argument. As an aside,
the concentration of blocker may have to be increased for cell lines above
the bone marrow protocol (but make sure that the percentage of dead cells
doesn't increase or you may be falsely removing the SP by killing the
cells!).

An additional influence on the shape of the SP is the excitation source - we
have seen this when comparing the Hoechst profiles of UV and violet excited
samples (Simpson et al " Out of the blue: a comparison of Hoechst side
population (SP) analysis of murine bone marrow using 325, 363 and 407 nm
excitation sources. " J Immunol Methods. 2006 Mar 20;310(1-2):171-81).
Speaking to people who are using the Aria they do say that you have to get
used to recognising a different shape!

Again with your scatter, you probably don't need log amplification (much of
what you are seeing is, I suspect, debris and noise) and the whole cells are
towards the upper right corner. However backgating the putative SP onto the
scatter plot, as you have done, is a useful exercise as you would expect the
SP cells to be fairly homogenous and form a tight scatter distribution.

Although they are using Hoechst in this paper, its another good one to look
at!
R Cabana et al. "The minimal instrumentation requirements for Hoechst side
population analysis: stem cell analysis on low-cost flow cytometry
platforms." Stem Cells. 2006 Nov;24(11):2573-81.

Good luck, it can be a minefield out there!
Derek


On 20/12/06 3:18 am, "Albert Tai" <acktai@exelixis.com> wrote:

> 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.

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Derek Davies					 Voice: (44) 020 7269 3394
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