Application of analytical cytology to current issues in the biomedical sciences

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Flow Cytometry Shared Resource Facility (FCU) initiatives Single Cell Sorting and PCR 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; Nguyen in the laboratory has explored several alternatives and our collaboration with a LMG postdoc, Peter Sykoa, has expanded into validating single cell qPCR with multiple primers with the goal to expand it to a translational project involving human organelle genetics. Comparative analysis of compensation strategies in flow cytometry 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. Bar-coding of human stem cells Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures. Boheler KR, Bhattacharya S, Kropp EM, Chuppa S, Riordan DR, Bausch-Fluck D, Burridge PW, Wu JC, Wersto RP, Chan GC, Rao S, Wollscheid B, Gundry RL. Stem Cell Reports 3:185-203, 2014