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. > > 1. First off, what is the best control to use for this two step > staining. I have tried both isotype-matched primary followed by labeled > secondary and just secondary with no primary. Which of these (or other) > is the best way to ascertain background fluorescence? > > 2. My cytokine antibodies are from R&D, my isotype-match control is from > PharMigen. Should I try to match suppliers for my isotype-match or is > this not a big deal. It means I have to spend a couple hundred more > dollars, which I don’t want to do, but I want to discern specific from > non-specific staining so I can trust my results. > > 3. I use btw 1.25 and 2.5 ug primary and 1 ug secondary per 1.5 million > PBMC. Does this concentration sound within acceptable ranges? I have > tried different concentrations, this appears to be the best range. Does > anyone know if surface or intracellular staining usually requires > different Ab concentrations? > > 4. Like I said, I work with human PBMC. I am looking at surface and > intracellular cytokines, and I have found a lot of variation between > donors. Some will respond to stimulus, some will not. Sometimes donors > have surface expression in freshly isolated PBMC, sometimes they don’t. > What degree of reproducibility is acceptable when staining PBMC? I can > see general trends, but I want to know how much leeway to allow these > trends. > > 5. I just tried to stain my cells for surface markers in PBS, 0.1% > sodium azide, but 1% human AB serum instead of fetal calf serum, hoping > this might better block non-specific interactions. Instead my cell > count was absurdly low, and there was tons of dead cells. Has anyone > seen this? Is this why one does not traditionally wash/stain cells in > human serum? > > I realize I have brought up a few different issues, so feel free to just > respond to individual issues, > > Thankyou in advance, > > Greg > > P.S. I enjoy eavesdropping on the flow discussions that take place in > this group, and I have found them very helpful on many occasions, so I > want to thank who ever is responsible for this group’s existence.
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:12 EST