Re: Absolute Quantification of Intracellular Cytokines

This is a very complex problem that has not yet been solved.  The  
primary issue is that no one has shown that the fluorescence intensity  
by ICS is linearly correlated with the amount of cytokine a cell may  
produce.  (And, in fact, it may not be -- remember that cytokine  
production measured for secreted cytokines is an integration of  
cytokine production over a long period, typically 24-72 hours; ICS is  
usually done over a 6-12 hour time frame.  Even if you do ICS in a 24  
hour stimulation, it's not clear that all of the cytokine produced and  
trapped in the cell remains equally accessible to antibodies to  
provide an integrated answer).

Then, there is the additional problem that the amount of cytokine  
produced in supernates comes from many cells.  So, to get an  
indication of how much cytokine might be produced in the culture, you  
would have to multiply the MFI (mean or median, take your pick) by the  
percentage of cells that are making the cytokine.  This accounts for  
situations where you have, for example, 1% of cells making an MFI of  
1000 versus another stimulation where 0.01% of cells making an MFI of  
10,000 -- the first culture will still make 10x as much cytokine in  
the medium (assuming everything is linearly related), even though it  
has 1/10th the cytokine per cell and 100 times the percentage of  
cytokine-producing cells!

To try to get at this, we developed the metric termed "iMFI" (for  
integrated MFI); see Darrah et al., Nat Med. 2007 Jul;13(7):843-50.   
The iMFI is simply the MFI x % of cells (% of the total cell  
population, not within CD4 or CD8, e.g.).  In fact, we showed that the  
iMFI was a predictor of vaccine efficacy in a mouse model for L. major  
infection -- in a case where the frequency of cytokine-producing cells  
did NOT correlate at all!

Loosely speaking, the iMFI correlated with ELISA done for cultures  
from similarly treated animals.  However, we have not yet done the  
correlations on the same cells, so I can't say with any certainty how  
well the iMFI correlates with ELISA.

In order to get decent MFI (and thus iMFI) measurements, you will need  
a well-calibrated instrument that gives you the same fluorescence  
every experiment (and your compensation better be spot-on).  That  
being said, I think we are still a long way from having standards that  
can be used in this application.

mr

On Feb 16, 2008, at 10:42 AM, Brian McFarlin wrote:

> Hello All,
>
> I have been scanning references in pubmed for an answer to my  
> subject line. It appears that the accepted way to report  
> intracellular cytokines is via MFI or number of positive cells. I  
> was curious of anyone is aware of a “standard” that you could run,  
> which would enable you to provide an absolute quantification (as a  
> pg/ml for instance). We typically run stimulations and measure  
> extracellular cytokine production with ELISA and it would be useful  
> to relate intracellular production back in the same units. I do not  
> know if this is even possible, but I suspect that it may be. For  
> instance I know that the multiplex bead arrays for flow involve a  
> standard curve. If anyone has any tips or suggestions, please let me  
> know. Thanks in advance,
>
> Brian
> -- 
> Brian K. McFarlin, PhD
> Assistant Professor Exercise Physiology / Nutrition
> University of Houston
> Department of Health and Human Performance
> 3855 Holman St.
> Houston, Texas 77204-6015
> (713) 743-9929 phone
> (713) 743-9860 fax