Optimized lymphocyte isolation methods for analysis of chemokine receptor expression.

Manipulations typically used to isolate enriched lymphocyte populations from peripheral blood can impact on the measured levels of chemokine receptors. Optimum sensitivity and accurate discrimination of receptor-expressing cell subsets therefore requires cell isolation methods that minimally affect expression levels. We used flow cytometry to examine the effects of different protocols for processing and staining T lymphocytes on chemokine receptor expression. Our results confirm that FACS analysis of some chemokine receptors is compromised after standard methods (such as Ficoll density separation). While the optimal method was typically to stain cells prior to lysing whole blood, this may not be practical in many experimental conditions. In general, we found that staining cells at 37C following Ficoll separation yielded excellent results. However, the precise method used will depend on which receptor is being measured. We used the optimal methods to compare the expression of chemokine receptors on naive and memory T-cell subsets using 8-color flow cytometry.