Flow Cytometry Shared Resource Facility (FCU) initiatives: Because of flow cytometry core responsibilities, these projects are on hold. Single Cell Sorting and PCR This project has been abandoned because of reproducibility issues.At best, we were achieving a 50% success rate for PCR sorted single cells. Initial summary: Based upon strong support from a user survey, the laboratory acquired the Advalytix (Ampligrid) technology specifically designed for RT- and qRT-PCR on single cells in 2010. From the flow cytometry (sorting) perspective, cells are sorted individually onto specially treated slides (which have microwells for each cell) and then processed for qRT-PCR on a benchtop unit designed for microscope slides. The core has been performing single-cell deposition into PCR plates sporadically for a number of users and this is a significant improvement of the technique. The product line was sold to Beckman Coulter who discontinued it in 2012. To remediate this problem, I identified potential replacements for the slides (from the original price of $50-75 each to $0.50), expanding the matrix for sorting onto 384 well slides, and finding replacement sources for the custom PCR reagents designed to work with the system. Core personnel (Wallace) has been validating the accuracy of sorting (something that has not been published before) and has documented identifiable problems with BD (sic Aria sorters) and the advantages of sorting on a MoFlo with narrower side stream angle of incidence and on the iCyt with 0 angle of incidence. The PCR arm has been problematic, particularly since the sealing solution is out of production and the formula still considered propriety. While users of the flow cytometry core have a broad range of expertise in this technology, we frequently have encountered situations where the design and implementation of their reagent panels is less than optimal. We realized that a paper documenting the proper strategies for selecting both compensation reagents and performing proper flow cytometric compensation for sample validation would benefit both our user base in the IRP as well as the flow cytometry community at large. We undertook a comprehensive analysis of typical compensation reagents, i.e. single color antibody controls using the same reagents in an antibody panels and expanded this to the use of CD8 and CD45 antibodies conjugated to each of the fluorochromes in the panel, and in collaboration with Spherotech Inc., the use of antigen-coated beads as compensation controls. This human-focused study also utilized a large number of antibody panels (12) that ranged from the simple (4-color), to 6- and 8-color, including panels that violated the common flow cytometric rules of antibody-fluorochrome selection, commercial panels, and the Euroflow panels used in the NIH trans-immunology imitative. Data acquisition is completed and an initial review of the results indicates there are a number of instances where the use of the same color antibodies (i.e. CD28 in the panel with use of CD28 as a compensation control) and either CD45 or CD8-matched fluorochrome controls are inappropriate. The results also show major differences in these beads among manufacturers adding to errors in selecting and setting compensation controls.
| Boheler, Kenneth R; Bhattacharya, Subarna; Kropp, Erin M et al. (2014) A human pluripotent stem cell surface N-glycoproteome resource reveals markers, extracellular epitopes, and drug targets. Stem Cell Reports 3:185-203 |
