Before I try to answer your questions, a question to you: Why are you using indirect (2 step) staining? There are superior directly labeled mAbs to a large number of cytokines. Are you looking at cytokines for which directly labeled reagents do not exist? > ---------- > From: Greg Neely > Sent: Thursday, March 11, 1999 15:18 > To: Cytometry Mailing List > Subject: Intracellular/extracellular cytokine detection using GAM-PE > secondary AB > > > > 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. > > > > > >
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