Decrease in MFI using HTS on LSRII

Jose Sancho jose.sancho at biogenidec.com
Fri Jun 8 15:01:02 EDT 2007


I found, FAcsCalibur, that the sample speed with the HTS affects the MFIs. 
 No changes from 1 to 1.5ul/s, above that there is significant decrease (2 
to 3 ul/s). 
Don't know about LSRII. I would try 1.5ul/s. I normally run at this flow 
rate.

Jose Sancho

Biogen Idec



Marty Bigos <mbigos at gladstone.ucsf.edu> 
07-Jun-2007 07:30 PM
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Re: Decrease in MFI using HTS on LSRII






I don't have a specific answer to your problems, but we have seen a 
number of related  problems with our HTS at various times. They have 
been sometime due to improper installation of the HTS by BD (it makes 
a difference where the HTS taps off the sheath line), and partially 
due to different running conditions (sample volume, sample flow rate, 
etc.). If you could give more specifics about those conditions, as 
well as your LSR laser conditions (especially window extension), that 
would help  in diagnosing your situation. Another tool is to look at 
fluorescence intensity vs time and see if the intensity is a flat line.

Marty Bigos, Director
Gladstone Flow Core

mail:
J. David Gladstone Institutes
1650 Owens Street
San Frnacisco CA 94158

Phones:
(office) 415-648-8346
(mobile) 415-845-8450

fax:
415-355-0855



On Jun 5, 2007, at 8:27 AM, Morschauser, Andrew wrote:

> Dear Group,
>
>
> I am posting the following question for a colleague:
>
>
> Here is my question for the Flow cytometry group.
>
>
>
> I have been trying to develop a plate based assay to measure a cell- 
> surface antigen on CHO cells using the LSRII-HTS system.
>
>
> I perform the staining in 12X75mm polypropylene tubes.  At the end 
> of the staining procedure, I transfer to polystyrene tubes and the 
> mean fluorescence of intensity is determined on the LSRII-HTS 
> system.  I then take that same sample, transfer to polypropylene or 
> polystyrene U-bottom 96-well plates and re-analyze the samples 
> using the HTS system.
>
>
> The MFI of the samples analyzed in the tubes is > than  the MFI of 
> the samples analyzed in the plates?  It is the same sample.
>
> Shouldn?t the MFI?s be relatively the same?
>
>
>  I would like this to be a plate-based assay because there are 
> quite a number of samples to be evaluated but I don?t feel 
> comfortable with the plate-based results.
>
>
> Any hints or suggestions would be greatly appreciated.
>
>
>
> Sincerely,
>
>
> Andy Morschauser
>
>



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