Decrease in MFI using HTS on LSRII

Ray Lannigan rlannigan at
Fri Jun 8 14:03:42 EDT 2007


		I would try this same test using beads instead of cells.
This would help isolate the instrument from the sample. Try using a
multi-peak hard dyed beads like Rainbow from Spherotech or FC3M from Duke
beads, and calculating the Q values for both tube and plate acquisitions .
Since the Q of a flow cytometer is how well it converts light from
fluorochrome molecules to photoelectrons, it is a pretty good way to assess
the sensitivity of your setup.

		Eric Chase, Bob Hoffman, and Jim Wood have published much on
this topic. I believe there was a recent addition to Current Protocols in
Cytometry instrumentation section (1.20) by Hoffman and Wood,
"Characterization of a Flow Cytometer  . 

A quick and easy way to evaluate Q is to just compare the CV's of a
moderately bright peak (peak 3 or 4 above the blank peak; Blank peak=1) in
the set of 8 peak Rainbow beads. The lower the CV the better the Q.  To get
an actual relative Q value use Q=1/CV2 * MEFL. The MEFL value can be
obtained from the bead manufacturer.  If the Q's for both conditions are
similar, look to the transfer conditions of your sample. If the Q's are
lower with the HTS setup I would guess there is a sample core misalignment
while injecting with the HTS and you might need a visit by a service


Raymond Lannigan

Cytek Development Inc.

Sales and Marketing Director


540-832-2794 (fax)

540-207-6709 (cell)

From: Morschauser, Andrew [mailto:andrew.morschauser at] 
Sent: Tuesday, June 05, 2007 11:28 AM
To: cyto-inbox
Subject: Decrease in MFI using HTS on LSRII

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.	


Andy Morschauser

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