All, I've had no luck in detecting the simplest cytokines in my frozen/thawed monocytes. The same samples, when fresh, produced IL-1b/TNF-a (LPS+BFA) and the T cells from the frozen/thawed experiments produced normal amounts of TNF-a/INF-g in response to PMA. Is there something extra about frozen monocytes that I need to be doing? I repeated this with 6 normals (fresh were run before freezing, frozen after 2 weeks at -80C using standard procedures) Fresh monos + LPS/BFA = 90% IL-1b (gated on 7aad-/CD33+ ,controls under 2%) Fresh monos + LPS/BFA = 99% TNF-a (gated on 7aad-/CD33+ , controls under 5%) Fresh Tcells +PMA/BFA = 40% TNF-a (gated on 7aad-/CD3+ ,controls under 1%) Fresh T cells+PMA/BFA = 26%INF-g (same as above) Frozen monos + LPS/BFA = 5% IL-1b (gated on 7aad-/CD33+) Frozen monos + LPS/BFA = 18% TNF-a (gated on 7aad-/CD33+) Frozen Tcells +PMA/BFA = 50% TNF-a (gated on 7aad-/CD3+) Frozen T cells+PMA/BFA = 24%INF-g (same as above) The reduction in monocyte production of IL-1b/TNF-a is also accompanied by a drop in the geometric mean of the cytokine positive events. Does anyone know how to bring the frozen monos' production up to match those of the fresh ? Maciej
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