Differential response of human basophil activation markers: a multi-parameter flow cytometry approach

<p>Abstract</p> <p>Background</p> <p>Basophils are circulating cells involved in hypersensitivity reactions and allergy but many aspects of their activation, including the sensitivity to external triggering factors and the molecular aspects of cell responses, are still to be focused. In this context, polychromatic flow cytometry (PFC) is a proper tool to investigate basophil function, as it allows to distinguish the expression of several membrane markers upon activation in multiple experimental conditions. </p> <p>Methods</p> <p>Cell suspensions were prepared from leukocyte buffy coat of K2-EDTA anticoagulated blood specimens; about 1500-2500 cellular events for each tested sample, gated in the lymphocyte CD45dim area and then electronically purified as HLADRnon expressing/CD123bright, were identified as basophilic cells. Basophil activation with fMLP, anti-IgE and calcium ionophore A23187 was evaluated by studying up-regulation of the indicated membrane markers with a two-laser six-color PFC protocol.</p> <p>Results</p> <p>Following stimulation, CD63, CD13, CD45 and the ectoenzyme CD203c up-regulated their membrane expression, while CD69 did not; CD63 expression occurred immediately (within 60 sec) but only in a minority of basophils, even at optimal agonist doses (in 33% and 14% of basophils, following fMLP and anti-IgE stimulation respectively). CD203c up-regulation occurred in the whole basophil population, even in CD63non expressing cells. Dose-dependence curves revealed CD203c as a more sensitive marker than CD63, in response to fMLP but not in response to anti-IgE and to calcium ionophore.</p> <p>Conclusion</p> <p>Use of polychromatic flow cytometry allowed efficient basophil electronic purification and identification of different behaviors of the major activation markers. The simultaneous use of two markers of activation and careful choice of activator are essential steps for reliable assessment of human basophil functions.</p

Inhibition of CD203c membrane up-regulation in human basophils by high dilutions of histamine: a controlled replication study

none5noPrevious research suggests that human basophil activation may be inhibited by histamine even at extremely low doses (high dilutions). In our experiment, membrane up-regulation of CD203c, which proved to be a more consistent activation marker than CD63, was significantly inhibited in samples treated with histamine at the dilutions of 2C, 12C, 14C, 15C and 16C. Control water dilutions/succussions did not show any significant effect. Therefore, using a strictly standardized flow cytometry protocol and a new dilution/succussion procedure, we have shown that low and high dilution of histamine do inhibit CD203c up-regulation in anti-IgE stimulated basophils.mixedS. Chirumbolo; M. Brizzi; R. Ortolani; A. Vella; P. BellaviteS. Chirumbolo; M. Brizzi; R. Ortolani; A. Vella; P. Bellavit