Decrease in MFI using HTS on LSRII

Laurie Lamoreaux llamorea at mail.nih.gov
Thu Jun 7 14:42:52 EDT 2007


Hi Andy,

You don¹t say what fluoro(s) you are staining with or how many samples you
have looked at.  Because you are splitting and transferring the sample after
it is stained, is it possible that the sample in the tube is exposed to
light long enough prior to transfer to plate wells to cause a decrease in
fluorescence of cells in the plate?

I would suggest repeating the experiment, splitting the sample(s) prior to
staining.  Stain in tubes and wells at the same time and protect from light
prior to sampling on the LSRII.  Also, is it possible for you to stain and
run in polystyrene tubes, eliminating the transfer step from polypropylene
into polystyrene?  

We routinely run pbmcs in 96-well v-bottom plates on our LSRII HTS and when
necessary have run identical samples in tubes and have not seen the same
phenomenon you have.

Laurie Lamoreaux
Immunology Laboratory
Vaccine Research Center, NIAID/NIH
40 Convent Dr., Rm. 3606
Bethesda, MD 20892
301-594-8633
llamorea at mail.nih.gov

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On 6/5/07 11:27 AM, "Morschauser, Andrew" <andrew.morschauser at roche.com>
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.
> Shouldn¹t the MFI¹s 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 don¹t feel comfortable with the plate-based
> results.
>  
> Any hints or suggestions would be greatly appreciated.
>  
>  
> Sincerely,
>  
> Andy Morschauser
> 


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