We have established a novel live-cell based indirect immunofluorescence assay for Paclitaxel based on PE fluorescence. Paclitaxel is primarily stored in the plant cell wall, and because our studies necessitate the sorting and selection of live cells both for mRNA expression analysis and re-culturing, it is critical that cells remain intact and not permeabilized or damaged throughout the staining procedure. This immunoassay was sufficient to stain respective amounts of cell wall-associated Paclitaxel in different sub-populations. Concomitantly, cells were also stained using fluorescein diacetate (FDA) to monitor viability. Multiparameter flow cytometry was performed and different Taxus cell populations were examined based on both PE and FITC fluorescence. To further validate the efficacy of this method, time-based viability tests were performed and demonstrated that cell viability remained high both during and after the staining procedure. We detect a broad range of Paclitaxel accumulation amongst Taxus cells in culture, providing an excellent target for fluorescence-activated cell sorting (FACS).
FACS can be effectively used to sort cells based on a variety of parameters including size, growth rate, protein production and metabolite production. Plant cells are challenging to sort due to their aggregated nature in suspension and relatively large size when compared to microbial and mammalian cells. We developed a new technique for high-throughput plant cell sorting using the BD FACSVantage equipped with a MacroSort option to accommodate our larger Taxus cells. A larger 200 μm nozzle with optimized optics and fluidics conditions was used in analysis and sorting of Taxus cells based on Paclitaxel accumulation. The instrument was calibrated with 25 micron beads and Taxus cells were first sorted on the basis of size. Following a successful (~80-90% purity) sort by size, cells were stained for Paclitaxel using the procedure mentioned above and sorted based on fluorescence. Cells were gated into two populations based on low and high Paclitaxel accumulation and sorted accordingly. We were able to attain a high level of purity (~90%) for both sorted populations, demonstrating that live Taxus cells can be effectively sorted according to Paclitaxel content. The new technologies developed here not only allow recovery of low and high accumulating cells for metabolic analysis, but also allow for re-culture and initiation of potentially superior cell lines with both enhanced Paclitaxel accumulation and stability.