PerFix-Expose1 and PerFix-nc1, innovative new reagent kits for intracellular staining from Beckman Coulter Life Sciences, minimize sample manipulation for a streamlined workflow that delivers high quality results. Developed for use on Beckman Coulter’s Gallios1 and Navios2 series flow cytometers and compatible with all Beckman Coulter Life Sciences’ fluorochromes, the kits enable a clear focus on the cytoplasmic and nuclear compartments of the cell.
PerFix-Expose (phospho-epitope exposure kit), which helps scientists identify and explore complex intracellular signaling pathways disrupted by cancer and other diseases, allows fluorochrome-coupled antibodies and other probes to enter into cells, their nuclei and other cell compartments, where they bind to proteins relevant to signaling pathways that have been disrupted by disease.
Cell fixation is accomplished using a cross-linking agent, while permeabilization of white blood cells and lysis of red blood cells is achieved using a detergent. A denaturing agent “unmasks” proteins bound to other proteins or DNA.
Beckman Coulter researchers developed the kit for use with whole blood, bone marrow or other fluids containing blood. Samples are fixed in small test tubes or deep-well microplates and the reagent is mixed with the cells. The kit can be used to study a variety of other intracellular and intranuclear antigens.
With PerFix-nc (no centrifugation), researchers can perform concomitant surface and intracellular staining for flow cytometry to take advantage of a new approach that provides easy access to the inside of human leucocytes. Analysis of multiple intracellular epitopes, or multiple intracellular markers along with cell surface markers, is accomplished by straightforward fixation, permeabilization and staining of leucocytes from peripheral whole blood, together with erythrocyte lysis.
The preparation is performed at room temperature, without the requirement for wash or centrifugation steps, and can be performed manually or automated on workstations such as the Beckman Coulter Life Sciences Biomek platform. PerFix-nc is flexible enough to allow washing if desired, and/or the use of a higher fixative concentration level, and makes intracellular staining as simple and efficient as surface staining.
PerFix-nc kit quality is characterized by mean fluorescence intensities and consistent performance in repeatability studies, as demonstrated by levels of cell recovery. The kit offers an original and powerful leucocyte permeabilization method followed by an optimized fixation step in a process that involves only 15 minutes of hands-on user time.
“More and more, flow cytometry researchers are focusing their studies towards the cell interior,” commented Mario Koksch, vice president and general manager of the Cytometry Business for Beckman Coulter Life Sciences. “With PerFix-nc, users can track down intracellular markers with ease, speed and accuracy, and combine this with cell surface labeling and analysis. PerFix-Expose helps scientists identify and explore complex intracellular signaling pathways disrupted by cancer and other diseases, which is essential for understanding and developing treatments.”
1 For Research Use Only; not for use in diagnostic procedures.
2 Pending clearance by the U.S. FDA. Not yet available for in vitro diagnostic use in the U.S.