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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