Effect of ex vivo storage on human peripheral blood neutrophil expression of CD11b and the stabilizing effects of Cyto-Chex.
CD11b is the alpha sub-unit of the CD11b/CD18 heterodimeric complex that has a key role in neutrophil-endothelial cell interactions and early events in inflammatory responses. Accurate assessment of CD11b expression by neutrophils can be problematic since the procedures that isolate cells from whole blood can increase antigen expression. This study used whole blood flow cytometry to monitor neutrophil CD11b expression following 0, 1, 2, 3 and 4 h of ex vivo incubation at room temperature and examined the effects of storage at 4 degrees C and the cell stabilization solution, Cyto-Chex, on antigen expression. Cyto-Chex-treated samples were also re-analyzed after 7 days storage at 4 degrees C. Neutrophil CD11b expression was high (> 90%) in five of the seven samples studied and incubation at room temperature induced a progressive upregulation which was significant after 2, 3, and 4 h (p < 0.05). Storage at 4 degrees C tempered this effect, although an increased expression (from baseline) was still observed after 4 h (p < 0.05). Cyto-Chex had no effect on the light scatter characteristics of treated cells and prevented CD11b upregulation in samples stored at room temperature. Furthermore, expression in Cyto-Chex-treated samples after 7 days was not significantly different from that observed in baseline (time 0) samples. In two samples demonstrating low neutrophil CD11b expression, a temperature-independent increase in the proportion of CD11b+ cells was observed over time. However, this increase was attenuated by treatment with Cyto-Chex. These findings indicate that sample storage significantly affects CD11b expression and caution should be exercised when interpreting data. Given that Cyto-Chex had no effect on the light scatter properties of cells and prevents antigen upregulation, this reagent may be useful for studies involving analysis of neutrophil activation antigen expression.
Sheffield Institute of Sports Medicine and Exercise Science, University of Sheffield, UK.
J M
JM
Saxton
A G
AG
Pockley
D001793:Blood Preservation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D016177:Macrophage-1 Antigen / Q000096:biosynthesis / Q000097:blood*; D008297:Male; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D012996:Solutions
D016177:Macrophage-1 Antigen; D012996:Solutions