BackgroundWe have developed a quantitative fluorescence cytometry (QFCM) method that can be used to measure BCL-2 family member proteins in cell lines and clinical samples. We described the validation of antibodies, methods development and application of the assay. MethodWe characterized and validated antibodies to BCL-2, BCL-X-L, and MCL-1 in cell lines to confirm specificity for flow cytometry. Each protein was measured in a panel of leukemia/lymphoma cell lines and B-cells from chronic lymphocytic leukemia (CLL) patients treated with the BCL-2/BCL-X-L inhibitor navitoclax. The cellular activity of various BCL-2 family member inhibitors alone and in combination was determined to demonstrate utility of our assay to correlate protein levels with efficacy. ResultsWe identified antibodies that were highly specific for each protein. The expression profile in cell lines as determined by molecules of equivalent soluble fluorochrome was comparable to western blot. Using our assay, BCL-2, BCL-X-L, and MCL-1 protein levels were shown to correlate with response to BCL-2 family inhibitors in vitro and could be measured in clinical samples. ConclusionsThis method can quantify BCL-2 family members in a specific, highly reproducible and sensitive fashion, and requires fewer cells compared to western blot. It is particularly useful for identifying BCL-2, BCL-X-L, and MCL-1 protein levels in a specific cell population within a heterogeneous population like those collected from CLL patients. These data show that our QFCM method can be used to facilitate the quantification and evaluation of biomarkers predictive of response in patients treated with BCL-2 family member inhibitors. (c) 2016 International Clinical Cytometry Society
