SP Cells

Derek Davies derek.davies at cancer.org.uk
Mon Nov 28 16:42:43 EST 2005


Hi Leonie,

A couple of comments. The blue filter you are using is fine for the 
Hoechst blue emission; however the red emission is far weaker and the 
bandpass you are using may be too narrow; I normally would have a 
long pass (620LP generally). Also the dichroic I use is normally a 
bit longer - 610LP (or Shortpass if you like, just change the 
channels - although saying that, it is also good to have a 
red-sensitive PMT for the Hoechst red emission). There is a degree of 
flexibility in the choice of filters, you can experiment to see which 
gives the best separation in your system.

The amplification of both the red and blue signals should be linear 
and as you are measuring different parts of the emission spectrum of 
the dye from a single excitation source, you don't need any 
compensation. There is more information on my website.

Good luck!
Derek


>Hi,
>I am having a bit of trouble setting up the Voltages and Compensation for
>SP Cells.
>I am acquiring the cells on a BD FACSVantage with DiVa option in Digital Mode.
>How critical are the filters,I have a Hoechst 33342 450/20 for the blue and
>a 660/20 for the red but I only have a 525DCSP for the splitter. Are these
>okay or not specific enough?
>
>I also have a problem in the two papers I am referring too. One paper
>states that the detectors should be in Linear mode while a second paper is
>in Log. Up till now I have been running in Log so which is correct and I
>would be very interested to hear anyones experiences.
>Thank you for your help.
>Best Wishes
>Leonie


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Derek Davies					Voice: (44) 020 7269 3394
FACS Laboratory,			FAX: (44) 020 7269 3479
London Research Institute,		e_mail: derek.davies at cancer.org.uk
Cancer Research UK		mobile: 07790 604112
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Web Page: http://science.cancerresearchuk.org/sci/facs/

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