Decrease in MFI using HTS on LSRII

Nebe-Von-Caron, G g.nebe-von-caron at unipath.com
Sat Jun 9 20:50:03 EDT 2007


Whilst not an expert on the LSR, from my limited experience with both
the LSR and the Aria I appreciate that they do something in the digital
processing between peak and area signals. I tried to understand how
particularly the biexponential scale changes automatically on the area
signals when I change the detector voltages. Eventually I worked on peak
signals. 
Can you check if the signals change in both peak and area signals? Also
do you have to checked the area scaling factor with the HTS module
running? If it changes the flow velocity of the sheath it might alter
the peak / area relationship as well.
Regards
Gerhard

________________________________

From: Laurie Lamoreaux [mailto:llamorea at mail.nih.gov]
Sent: Thu 07/06/2007 19:42
To: cyto-inbox
Subject: Re: Decrease in MFI using HTS on LSRII


Hi Andy,

You don't 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.
	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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