Gfp Compatibility with Edu Cell Proliferation Assay

Abstract Examining cell proliferation in GFP expressing cells is of general interest in many aspects of biology including regenerative medicine, stem cells, developmental biology and some fields of cancer research. However, visualization of GFP expression is not readily compatible with commonly used proliferation assays which incorporate a thymidine analog to directly measure S-phase proliferation of DNA. With the BrdU (bromo-deoxyuridine) assay, an antibody based detection method, alcohol for fixation and hydrochloric acid for DNA denaturation is commonly used. Neither chemical is compatible with GFP fluorescence. For imaging applications there are some methods to avoid the use of HCl as a denaturant with the BrdU assay, however; they are typically “home-brew” methods used to partially digest the DNA, adding extra steps, and not easily performed. The much faster and reliable EdU (ethynyl-deoxyuridine) cell proliferation assay which uses click chemistry for detection of S-phase proliferation of DNA, uses formaldehyde based fixation and avoids the use of HCl for DNA denaturation. However, the use of copper to catalyse the click reaction also negatively affects GFP fluorescence. Anti-GFP antibodies can be used in the click reaction work flow, to “retrieve” the lost GFP fluorescence but are not convenient and add extra steps. We present recent improvements to the click chemistry based EdU cell proliferation assay which minimizes the loss of GFP and other fluorescent proteins signals and avoids the need for “work around” methods. The resulting click reaction is both more rapid and brighter than the “classic” click EdU assay. The modifications preserve most of the GFP fluorescence and permit multiplex detection with EdU with no change in the work flow of the classic click EdU assay. We optimized components in the improved click reaction conditions and tested compatibility with various fluorescent proteins. Examples of click chemistry and GFP/RFP/mCherry compatibility are presented using the EdU cell proliferation assay in both cell culture and in GFP expressing tissue. Additionally, improvements are demonstrated in with other applications of click chemistry assays used for imaging as well as with on flow cytometry platforms where GFP, R-phycoerythrin (R-PE), or other fluorescent proteins are commonly combined with cell proliferation assays. The use of the modified click reaction is an enabling improvement over originally described copper based click reactions and will further enhance the utility of EdU based cell proliferation. Citation Format: Scott T. Clarke, Zhichao Song, Kelvin Y. Kwan, Carolyn DeMarco, Aleksey Rukavishnikov, Upinder Singh, Kyle Gee. GFP compatibility with EdU cell proliferation assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5098. doi:10.1158/1538-7445.AM2014-5098