Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry

Background: Measurement of cell proliferation via BrdU incorporation in combination with multicolor cell surface staining would facilitate studies on cell subsets that require multiple markers for their identification. However, the extent to which the often harsh cell preparation procedures required affect the staining quality of more recently developed fluorescent dyes has not been assessed. Methods: Three cell preparation protocols for BrdU measurement were compared for their ability to maintain fluorescent surface staining and scatter parameters of in vivo BrdU-labeled cells by flow cytometry. A 10-color fluorescent panel was developed to test the quality of surface staining, following cell treatment and the ability to perform BrdU measurements on even small B lymphocyte subsets. Results: All cell preparation procedures affected the quality of fluorescent and/or scatter parameters to varying degrees. Paraformaldehyde/saponin-based procedures preserved sufficient fluorescent surface staining to determine BrdU incorporation rates among all splenic B cell subsets, including B-1a cells, which constitute roughly 0.5% of cells. Turnover rates of B-1a cells were similar to immature B cells and higher than those of the other mature B cell subsets. Conclusion: Paraformaldehyde/saponin-based cell preparation procedures facilitate detailed cell turnover studies on small cell subsets in vivo, revealing new functional information on rare cell populations.