Online Purchasing Account You are logged on as Guest. LoginRegister a New AccountShopping cart (Empty)
United States 

Flow Cytometry Revealed: Intercellularly Transferred P-glycoprotein in the Human Blood-Brain Barrier is Active

Posted By Jan-Henning Martens
Tags: Cancer, Drug Discovery, Neuroscience

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.

Share this TechNote

Never miss a new TechNote!

Receive our TechNotes as soon as they are published.


Follow Us!

 
comments powered by Disqus

References:

  1. 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.
  2. A. Levchenko, et al. Intercellular transfer of P-glycoprotein mediates acquired multidrug resistance in tumor cells. Proc. Natl. Acad. Sci. U S A. (2005) 102: 1933.

Related Products

HSP70/HSP72 monoclonal antibody (C92F3A-5) (FITC conjugate) 

Flow Cytometry | Print as PDF
 
ADI-SPA-810FI-D 50 µg 219.00 USD
 
ADI-SPA-810FI-F 200 µg 430.00 USD
Do you need bulk/larger quantities?
 

HSC70/HSP73 monoclonal antibody (1B5) (PE conjugate) 

Flow Cytometry | Print as PDF
 
ADI-SPA-815PE-E 100 µg 330.00 USD
Do you need bulk/larger quantities?
 

HSC70/HSP70 monoclonal antibody (N27F3-4) (DyLight™ 488 conjugate) 

Flow Cytometry | Print as PDF
 
ADI-SPA-820-488-E 100 µg 327.00 USD
Do you need bulk/larger quantities?
 

E-cadherin monoclonal antibody (HECD-1) 

Obtained by fusing the P3-X63-Ag8-U1 myeloma cell line with splenocytes of BALB/c mouse immunized with human breast tumor cell line MCF-7., ELISA, Flow Cytometry, ICC, IHC (FS), IHC (PS), WB, FUNC | Print as PDF
 
ALX-804-201-C100 100 µg 690.00 USD
Do you need bulk/larger quantities?
 

CD14 (human) monoclonal antibody (biG 2/RoMo-1) 

ELISA, Flow Cytometry, IP | Print as PDF
 
ALX-804-498-C100 100 µg 258.00 USD
Do you need bulk/larger quantities?
 

CD3 (human) monoclonal antibody (MEM-57) 

Flow Cytometry, IP, FUNC | Print as PDF
 
ALX-805-057-C100 100 µg 194.00 USD
Do you need bulk/larger quantities?
 

p53 (human) monoclonal antibody (DO-7) 

ELISA, Flow Cytometry, IHC, IP, WB | Print as PDF
 
BML-PW1095-0025 25 µg 184.00 USD
Do you need bulk/larger quantities?
 

HIF-1α monoclonal antibody (H1α67) 

ELISA, Flow Cytometry, ICC, IF, IHC, IHC (FS), IHC (PS), IP, WB | Print as PDF
 
BML-SA287-0100 100 µl 367.00 USD
Do you need bulk/larger quantities?
 

Bak (human) monoclonal antibody (TC100) 

Flow Cytometry, ICC, WB | Print as PDF
 
BML-SA298-0050 50 µg 273.00 USD
Do you need bulk/larger quantities?
 

GFP-CERTIFIED® Apoptosis/Necrosis detection kit 

Multiplex assay that distinguishes between  healthy, early apoptotic, late apoptotic and necrotic cells, compatible with GFP and other green fluorescent probes
Flow Cytometry, Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51002-25 25 assays 157.00 USD
 
ENZ-51002-100 100 assays 371.00 USD
Do you need bulk/larger quantities?
 

TOTAL-NUCLEAR-ID® green/red nucleolar/nuclear detection kit  

Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51006-500 1 Kit 245.00 USD
Do you need bulk/larger quantities?
 

NUCLEAR-ID® Red cell cycle kit (GFP-CERTIFIED®) 

Convenient kit for studying cell cycle progression by various applications
Flow Cytometry, Fluorescence microscopy | Print as PDF
 
ENZ-51008-100 1 Kit 321.00 USD
Do you need bulk/larger quantities?
 

ROS-ID® Total ROS/Superoxide detection kit 

Directly Monitor Global Levels  of Reactive Oxygen Species (ROS), and Specifically Superoxide
Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51010 1 Kit 235.00 USD
Do you need bulk/larger quantities?
 

ROS-ID® Total ROS detection kit 

Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51011 1 Kit 172.00 USD
Do you need bulk/larger quantities?
 

ROS-ID® Superoxide detection kit 

Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51012 1 Kit 172.00 USD
Do you need bulk/larger quantities?
 

NUCLEAR-ID® Green cell cycle kit  

Cell Cycle Results Independent of Incubation Time, Temperature, Dye and Cell Concentrations
Flow Cytometry, Fluorescence microscopy | Print as PDF
 
ENZ-51014-100 1 Kit 312.00 USD
Do you need bulk/larger quantities?
 

MITO-ID® Membrane potential detection kit  

The only assay that monitors energetic status using a simple mix-and-read, no-wash protocol
Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51018-0025 25 tests 161.00 USD
 
ENZ-51018-K100 100 tests 462.00 USD
Do you need bulk/larger quantities?
 

NUCLEAR-ID® Green chromatin condensation detection kit  

A highly permeable green-emitting  dye for enhanced detection of apoptosis-induced chromatin condensation
Flow Cytometry, Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51021-K200 1 Kit 284.00 USD
Do you need bulk/larger quantities?
 

EFLUXX-ID® Green multidrug resistance assay kit 

Simple No-wash Assay for  Simultaneous Monitoring of All 3 Major ABC Transporter Proteins – MDR, BCRP & MRP
Flow Cytometry, Fluorescence microscopy | Print as PDF
 
ENZ-51029-K100 1 Kit 538.00 USD
Do you need bulk/larger quantities?
 

EFLUXX-ID® Gold multidrug resistance assay kit 

Simple no-wash assays for simultaneous monitoring of all 3 major  ABC transporter proteins – MDR, BCRP & MRP
Flow Cytometry, Fluorescence microscopy | Print as PDF
 
ENZ-51030-K100 1 Kit 538.00 USD
Do you need bulk/larger quantities?
 

CYTO-ID® Autophagy detection kit 

A no-transfection, quantitative assay for monitoring autophagy in live cells
Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51031-0050 50 tests 205.00 USD
 
ENZ-51031-K200 200 tests 420.00 USD
Do you need bulk/larger quantities?
 

PROTEOSTAT® Aggresome detection kit  

Robust, quantitative detection of aggresomes relevant to the study of neurodegenerative disease, liver disease and  toxicology
Flow Cytometry, Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51035-0025 25 tests 108.00 USD
 
ENZ-51035-K100 100 tests 294.00 USD
Do you need bulk/larger quantities?
 

CYTO-ID® Green long-term cell tracer kit 

Live cell fluorescent labeling over extended time periods with no apparent toxic effects
Flow Cytometry | Print as PDF
 
ENZ-51036-K025 1 Kit 204.00 USD
Do you need bulk/larger quantities?
 

CYTO-ID® Red long-term cell tracer kit  

Live  cell fluorescent labeling over extended time periods with no apparent toxic effects
Flow Cytometry, Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51037-K025 1 Kit 195.00 USD
Do you need bulk/larger quantities?
 

ROS-ID® Hypoxia/Oxidative stress detection kit 

Specific detection system for simultaneous analysis of hypoxia and oxidative stress using microscopy and flow cytometry
Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS | Print as PDF
 
ENZ-51042-0125 125 tests 105.00 USD
 
ENZ-51042-K500 500 tests 367.00 USD
Do you need bulk/larger quantities?
 

FLOWSCRIPT® HPV E6/E7 assay kit 

Flow cytometry-based assay for the single cell detection of mRNAs for HPV oncoproteins E6 and E7
Flow Cytometry, Fluorescent detection | Print as PDF
 
ENZ-GEN300-0100 100 tests Inquire for pricing
Do you need bulk/larger quantities?
 

Cellular senescence live cell analysis assay 

Simple assay to quantify senescence-associated β-galactosidase activity in live cells using flow cytometry
Flow Cytometry, Fluorescent detection | Print as PDF
 
ENZ-KIT130-0010 10 assays 293.00 USD
Do you need bulk/larger quantities?
 

Recommend this page

 
For Research Use Only. Not for use in diagnostic procedures.
Keep in touch

©2017 Enzo Life Sciences, Inc.,