Flow cytometry is a powerful technology for investigating many aspects of cell biology and for isolating cells of interest. Flow cytometry utilizes highly focused, extremely bright beams of light (usually from lasers) to directly reveal aspects of cells - e.g. size and granularity - by the way light is scattered, or indirectly by introducing fluorescent probes to cell compartments, e.g., through DNA binding dyes that stain nuclei or by fluorescently labeled antibodies that specifically detect cellular proteins. The power of flow cytometry derives from the fact that it quantitatively analyzes individual cells, thus permitting the identification of subpopulations within a sample. The power of single cell analysis is compounded by the ability to measure multiple parameters simultaneously on each individual cell, to do this very fast (in excess of 20,000 cells/second), and to isolate/purify/sort desired subpopulations (up to 4 simultaneously).
Multidrug resistance (MDR)
One of the many recent applications of flow cytometry is the cellular analysis of multidrug resistance (MDR) for treatment of diseases such as cancer. The mechanism that is most commonly encountered in MDR is the increased efflux of a broad class of hydrophobic cytotoxic drugs that is mediated by one of a family of energy-dependent transporters, known as ATP-binding cassette (ABC) transporters. First described in the 1970s, several members of the ABC transporter family, such as P-glycoprotein (Pgp, also known as MDR1 or ABCB1), MRP1-5, and BCRP can induce MDR. The broad substrate specificity and the abundance of ABC transporter proteins might explain the difficulties faced during the past 30 years in attempting to circumvent ABC-mediated MDR in vivo. Drug resistance is not only a problem in cancer treatment, but also in several other diseases, including brain disorders such as epilepsy or depression. For example, overexpression of Pgp at the blood-brain barrier (BBB) is discussed as a major mechanism of pharmacoresistance in such disorders. In addition, about ten years ago, Andre Levchenko and colleagues from Memorial Sloan–Kettering Cancer Center in New York reported that intercellular transfer of the efflux transporter Pgp can mediate acquired MDR. Intercellular transfer of proteins is an integral part of communication between cells, involving mechanisms such as tunneling nanotubes that bridge neighboring cells or the release and binding of protein-containing membrane microparticles and extracellular vesicles. The capability of cells to acquire MDR by intercellular transfer of efflux transporters such as Pgp added another dimension to the ways cells can acquire a particular cell surface protein-mediated phenotype and since then, numerous studies have confirmed these findings and explored mechanisms involved in this transfer.
Intercellular Pgp transfer
In their very recent study, Andreas Noack and colleagues from the University of Veterinary Medicine and the Medical School of Hannover, Germany, investigated whether intercellular Pgp transfer as reported for cancer cells is also a physiological defense mechanism of brain capillary endothelial cells that form the BBB. For this study they co-cultured human brain capillary endothelial hCMEC/D3 cells (Pgp-recipient cells) with an equal number of hCMEC/D3-MDR1-EGFP cells (Pgp-donor cells). Using this approach, they were able to observe intercellular Pgp-EGFP transfer from donor to recipient cells that occurred by either direct cell-to-cell contact between adherent cells or by Pgp-EGFP enriched vesicles that were exocytosed by Pgp-EGFP donor cells and endocytosed by adherent Pgp-EGFP recipient cells. To address the question whether the transferred Pgp-EGFP fusion protein was functional flow cytometry experiments were performed utilizing Enzo’s EFLUXX-ID® Gold multidrug resistance assay kit,in combination with the Pgp inhibitors tariquidar and verapamil. These assays showed that the Pgp-EGFP fusion protein in the donor cells was indeed functional in the co-cultures and that the recipient cells to which Pgp-EGFP had been transferred gained a significant elevation of Pgp activity by a factor of 1.5 in comparison to wildtype cells. Additional experiments with the major antiepileptic drug valproate and the HDAC inhibitor trichostatin A revealed that both drug treatments increased Pgp-EGFP transfer and that this increase was associated with enhanced acetylation of histone H4. The findings of this study present a novel mechanism of alterations in BBB functionality by non-genetic transfer of a resistance phenotype that might have important implications for BBB functioning and resistance to therapy.
From our complex CELLESTIAL® portfolio of fluorescent probes and assay kits for cellular analysis to our large portfolio of antibodies validated in flow cytometry, Enzo Life Sciences provides a complete set of flow cytometry tools, some of which are described below.
A. Noack, et al. Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors. Sci. Rep. (2016) 6: 29253.
Multiplex assay that distinguishes between healthy, early apoptotic, late apoptotic and necrotic cells, compatible with GFP and other fluorescent probes (blue or cyan)
Flow Cytometry, Fluorescence microscopy, Fluorescent detection | Print as PDF