Greg Behbehani, a physician scientist, is focused on translational research, leveraging his work treating patients in clinical trials for high-risk hematologic malignancies towards the ultimate goal of utilizing data generated in the laboratory to predict ideal chemotherapy regimens and optimal novel agents for individual patients.
Most recently Dr. Behbehani has contributed to a published paper surrounding research of new protocols for barcoding on mass and fluorescence cytometry . The dataset is hosted and publicly available on Community Cytobank; a free resource for individuals interested in the analysis, workflow, and protocols represented in this paper.
Transient Partial Permeabilization with Saponin Enables Cellular Barcoding Prior to Surface Marker Staining.
Fluorescent cellular barcoding (FCB) and mass-tag cellular barcoding (MCB) methods can be used to covalently stain cell populations with either fluorescent or mass tags to allow simultaneous antibody staining and cytometric analysis of several samples in a single tube. These techniques enable high throughput analysis of multiple samples while minimizing inter-sample subjectivity due to procedural limitations (sample processing and instrument variation), and reduce reagent consumption. Current methods for FCB and MCB preform dye or mass-tag labeling after cell permeabilization with alcohol; however, alcohol permeabilization can disrupt surface marker epitopes, necessitating that the surface staining be performed prior to barcoding or that surface marker assessment be limited to those epitopes that are not disrupted by alcohol fixation.
The method we have developed employs transient partial cell permeabilization of fixed cells using a low concentration (0.02%) of saponin. This transient partial permeabilization allows for barcode labeling and accurate surface marker staining without significant interference due to staining of intracellular epitopes (that might lead to incorrect immunophenotypic profiles). We have demonstrated that this partial permeabilization step greatly increases the efficiency and decreases the variability of barcode staining using either fluorescent or mass-tag reagents. We have also confirmed that this approach does not allow staining of intracellular signaling proteins or intracellular pools of surface markers during the surface staining step. To validate this approach, we performed a 27-marker stain of normal human bone marrow that demonstrated equivalent staining across a broad collection of surface markers and immunophenotypic cell types. Lastly, we demonstrated the utility of this approach for performing direct comparisons of surface marker expression between four human peripheral blood samples. This method simplifies the process of barcoding samples when surface marker characterization is required and allows for extremely accurate comparison of surface marker expression levels among samples within a given experiment.
To find out more about Dr. Greg Behbehani and his research, read his Cytobank user story located here.