Bob, I take exception to your use of the term "cookbook permeabilization" and the "notoriously variable results" that result. In my experience, the true "cookbookers" are the investigators that rely on the often overpriced, oversimplified commercially available fixation/permeabilization kits, because they must blindly rely on the "cookbook" you give them. Presumably, they do not have the time, motivation, imagination or skill to master an assay. There are a lot of excellent papers (JEM, Nature, Science, J.I.) out there using what you term "cookbook" methods. I will be happy to go hand to hand at the upcoming FASEB meeting with any of your technical staff examining Ag specific responses by flow and will bet a case of Champagne (OK, 1/2 a case) that my "notoriously variable cookbook" system will have less noise and as good a signal as the out of the box "fix and perm" kit from Caltag. Are we on? Kevin Holmes can be the referee, results to reported to the Cytometry mailing list. In all seriousness (although the above offer stands!) I think your comments below are one sided and not supported by fact. Sure, a lot of people have problems with the home made reagents, but honestly, do you think that is not the case with your kits? Have you ever followed up with a random 100 purchasers to gauge the success rate? The bottom line is that more than one approach may work, and different investigators have different needs. Ultimately what counts is the veracity, quality and substance of the data and that can be achieved with either approach. > _______________________ > Calman Prussin, M.D. > Head, Clinical Allergy and Immunology Unit > Laboratory of Allergic Diseases > NIAID/ National Institutes of Health > > > ---------- > From: Bob Johnson > Sent: Sunday, March 14, 1999 12:50 PM > To: Cytometry Mailing List > Subject: Re: Intracellular/extracellular cytokine detection using > GAM-PE secondary AB > > > Re: Intracellular Cytokine Assays > Hi Greg, > I am Bob Johnson from Caltag Laboratories (sold in Canada through > Cedarlane). We have considerable experience in intracellular assays and > we > introduced the first commercial permeabilization system (Fix and Perm). > I > can mention a few pitfalls and suggestions. > > 1. First, you don't mention your permeabilization system. Cookbook > methods just using saponin give notoriously variable results. You > should > use Fix & Perm or a comparable, commercial system from another > manufacturer > 2. Unconjugated cytokine antibodies from R&D may not be the right > choice > for flow cytometry. Chances are they were chosen because they work in > ELISA. Clones have to be specifically chosen for flow cytometry and the > best choice is always a direct fluorochrome conjugate. > 3. Normally, the use of unconjugated antibodies with a second step is > to > be avoided for intracellular assays. Backgrounds are higher and > considerable experimentation is required to balance the amounts of > primary > and secondary antibody. > 4. The expression of certain intracellular cytokines such as IL-4 is > too > low for the use of the FITC fluorochrome. You need a PE antibody. If > you > rely on cookbook permeabilization methods and an FITC second antibody, > your > chances of success are minimal. If you use these same permeabilization > methods with a PE second antibody, they are not improved much. PE is a > big > molecule and the cookbook permeabilization methods rarely work reliably > with > even a direct PE. > 5. If you are blocking, I suggest the use of IgG from the host animal > of > the antibody ..e.g. a mouse anti-human monoclonal, use mouse IgG. > > The bottom line is you will have difficulty getting reproducible results > unless you use a commercial permeabilization system and directly > conjugated > monoclonal antibodies which are proven in flow cytometry. > Regards > Bob Johnson > > Greg Neely wrote: > > > Hello, My name is Greg, I am a graduate student at the university of > > Calgary, Canada. > > > > I do intracellular/extracellular flow cytometry for cytokines in human > > PBMC. I do not have fluorochrome-labeled cytokine-specific antibodies, > > so I use a two-step process. I have a few questions about how I have > > been doing things, and nobody around here can give me good answers, so I > > thought some of you may be able to help. > > >
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