[Cytometry] Antibody master mix question

Mario Roederer roederer at drmr.com
Mon Jan 30 08:06:18 EST 2012


We have observed no issues with combining ANY tandems.  I would be curious to see any data that shows a problem -- please ask your vendor "specialist" to share such data (please, only real data, not hearsay) and circulate it!

Antibody titres are based only on antibody concentration when cell staining.  For each antibody, calculate the final antibody concentration in your titration experiment that gave optimal results, and then make your master mix accordingly.  It's irrelevant how many other antibodies are present, or how much you dilute your master mix prior to use, as long as the FINAL antibody concentration is correct.  Don't get caught up with intermediate dilutions or other additions etc. -- the only thing you need to figure out is how much of each antibody ends up with the cells -- make that correct.

mr


On Jan 25, 2012, at 1:58 PM, Suter, Mike wrote:

> This is an interesting and timely topic!    We are launching into 10 color clinical flow cytometry, and I had a couple of questions:
> 
> I've heard (from a vendor applications specialist) that some tandems don't cocktail well together, such as the APC-AA700 and APC-AA750.   Does anyone have experience with decreased stability or adverse antibody interactions with certain fluorochromes over time, which would require omitting those from a cocktail?  
> 
> I've also heard different stories on whether the amount of ab used in titration needs to take into account the diluting effect of other ab's.  For instance:  if you titer an antibody in a 2 color tube, then use it in a 10 color experiment, the final dilution is 5X that in the original titration tube.  The number of fluorochrome molecules in the tube is the same, but they are less concentrated/more spatially separated.   Is it sufficient to extend the incubation to achieve saturation, or should the final dilution be factored into the titration?   I'm guessing the former is true, and Rob Sutherland's comment that he brings his cocktail volumes up to a uniform final volume with buffer implies that works.   However I've heard both sides argued.
> 
> Thanks,    Mike
> 
> Michael Suter, MT (ASCP) SH, ICEE
> 
> msuter at peacehealthlabs.org
> www.peacehealthlabs.org
> 123 International Way, Springfield, OR 97477
> 
> 
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