This is in contrast to CyTOF data, which requires antibodies to be conjugated to lanthanides and optimized by the individual investigator, and for which a simple and straightforward data analysis method is yet to be devised. The use of well-established and validated commercially available antibodies means high quality protein-level data is obtained that will have a very high validation rate; indeed flow cytometry is a platform that is commonly used as the means of validation for other high-throughput methods such as gene expression profiling or mass spectrometry. Thus this platform combines the advantages of a highthroughput screen with a SCH772984 ERK inhibitor detection method that is sensitive and highly reproducible, as demonstrated by the high correlation coefficients obtained between replicate runs. Furthermore, the use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies and/or development of more focused panels for specific applications such as 11-plex flow cytometry and/or CyTOF. Notably, only 5 out of 363 antibodies were found to be potentially problematic, in that they did not stain cell types that are reported to express the corresponding antigens, supporting the robustness of this approach. A requirement for FC analysis is that cells must be in single cell suspension, thus cultured adherent cells are detached by trypsinization and solid tumors are enzymatically dissociated. In addition it is common to use cryopreserved cells or to fix cells prior to analysis. We were concerned that these experimental manipulations would lead to significant changes in cell surface antigen detection. It is reassuring that detection of the majority of antigens assayed was stable; however detection of a number of markers was significantly altered. For the purposes of this study we have chosen to define a significant change as at least 5% absolute change and at least a two-fold change in relative detection. With that definition, eight antigens were significantly altered by fixation after staining, though most often to reduce the mean fluorescence intensity rather than the overall pattern of antigen detection. Nine antigens were altered by cryopreservation and thawing; one antigen, CD138 was reduced, while the remainder were increased. The latter are almost exclusively expressed on lymphoid cells, and we hypothesize that these changes in apparent antigen detection may be accounted for by differential ability of different cell types in the pool to survive the cryopreservation and thawing process. Finally, 22 antigens were significantly altered by enzymatic digestion. All enzymes caused some change; trypsin, dispase and collagenase caused the most changes and are known proteases, but hyaluronidase and DNase also caused some alterations in antigen detection, though we cannot exclude impurity in the cell culture grade products used in routine practice in this setting. Of the 22 antigens showing significant change, the majority are reported to have extracellular proteolytic cleavage sites, which in some cases may be integral to the mechanism of action of the molecule. While the majority of influenced antigens showed decrease with enzymatic digestion