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R + Cu(I) Miguel Garcia 1, Laetitia Roh 1, Jessica Sordet-Dessimoz 2, Gian-Filippo Mancini 2, Scott Grecian 3, Upinder Singh 3, Kyle Gee 3, Scott T. Clarke 3 1 Flow Cytometry, Ecole Polytechnique Fédérale De Lausanne, 2 Histology Core Facility, Ecole Polytechnique Fédérale De Lausanne, 3 Cellular Imaging, Molecular Probes/Life Molecular Probes ® Labeling and Detection Technologies – Life Technologies, 29851 Willow Creek Road, Eugene, Oregon 97402 USA ABSTRACT EdU (ethynyl-deoxyuridine) incorporation into nascent DNA detected with copper catalyzed click chemistry (CuAAC) as a method for measuring cell proliferation was first described in 2008 1,2. This method reduces the assay time and improves the work flow compared to the traditional method using antibody based BrdU detection. However, the presence of copper and reactive oxygen species (ROS) mediated damage to fluorescent proteins prevents the simultaneous detection of EdU and GFP fluorescence. We present here chemical modifications to the click reaction resulting in “copper safe” catalysis of the click reaction, whereby GFP fluorescence is preserved while EdU based cell proliferation is detected. The basis of the improvement is the use of a copper (I) ligand combined with a modified dye azide detection reagent. Together, the copper ion is sequestered with the ligand and prevented from damaging GFP fluorescence but still remains available to catalyze the click reaction. Ligands with excessively high affinity for copper protect GFP fluorescence, but result in reduced click reaction. The modified dye azide with an intrinsic affinity for copper reduces ROS damage and facilitates the click reaction. We used 4% paraformaldehyde fixed OCT embedded tissue from a mouse expressing beta-actin-GFP pulsed with 50 mg/kg body weight EdU four hours prior to sacrifice to optimize the click reaction conditions which best preserved the GFP fluorescence while obtaining a bright EdU signal. In cultured cells, we used GFP expressing cell lines which were labeled with EdU, then analyzed by high content imaging (HCA) and flow cytometry. Both imaging and flow cytometry data are presented showing simultaneous detection of EdU based cell proliferation with GFP fluorescence. The use of the described modified click reaction is an enabling improvement over earlier described click reactions and will further enhance the utility of EdU based cell proliferation assays. INTRODUCTION Click chemistry is a copper catalyzed covalent reaction between an alkyne and an azide used to label biological macromolecules like DNA, RNA and protein. Copper used in the reaction causes unwanted side reactions such as quenching of GFP fluorescence. Use of a copper chelate can protection GFP but inhibit the click reaction. Use of a modified azide dye improves the click reaction efficiency and helps to chelate copper Use of an optimized amount of copper chelate combined with modified dye azide creates an ideal click reaction with rapid reactivity and protective conditions for GFP fluorescence CONCLUSIONS Achieving simultaneous bright click reaction and GFP fluorescence requires modifying the click reaction with an optimal amount of a copper chelate Use of copper chelate does not diminish the sensitivity of detection of EdU in tissue The use of modified azide dye increases the click signal compared to the use of the unmodified dye azide in the click reaction Combining modified dye azide with a copper chelate is a clear improvement to the biological applications of click chemistry Improved click chemistry for EdU cell proliferation combined with GFP fluorescence Life Technologies 5791 Van Allen Way Carlsbad, CA 92008 www.lifetechnologies.com RESULTS Figure 1: Improving Click Chemistry Figure 4: All EdU positive cells are detected with 2X copper chelate Figure 7 : Use of modified azide dye in the click reaction along with the copper chelate protects GFP fluorescence from quenching during the click reaction. U-2 OS cells transduced with CellLight™ Histone 2B-GFP BacMam 2.0 were pulsed with 10 M EdU for 2 hours prior to fix/perm and click labeling then stained for DNA content using FxCycle™ Violet (DAPI). The optimal amount of chelate is achieved with a balance between obtaining a bright click reaction and protecting GFP fluorescence. Increasing the chelate amount diminishes the click signal while protecting GFP fluorescence. For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Figure 1 : Click chemistry: Cu(I) catalyzed cycloaddition between an azide and an alkyne results in covalent bond of a dye to a tagged macromolecule (a). Addition of a copper chelate and use of modified azide dye to co-ordinate copper creates faster click reaction that has improved biological compatibility 3. When EdU is incorporated within the cellular DNA, cell proliferation rates (S-phase fraction) can be determined from the proportion of cells replicating their DNA which label with the click reaction (b). Figure 2: Modified click chemistry is GFP and phalloidin compatibility Figure 2 : Multi-channel fluorescent image of nascent RNA labeled with ethynyl uridine (EU) and detected with modified click chemistry. A375 melanoma cells expressing Erk2-GFP protein were pulsed with 200 mM EU for 90 minutes. Click labeling using copper chelate shows improved copper compatibility with GFP (c) and phalloidin (d) staining. The fluorescent signal was preserved while detecting click specific signal (e). REFERENCES 1.A. Salic, T. J. Mitchison, Proc Natl Acad Sci U S A 105, 2415 (Feb 19, 2008) 2.S. B. Buck et al., Biotechniques 44, 927 (Jun, 2008). 3.C. Uttamapinant et al., Angew Chem Int Ed Engl, (May 3, 2012). a bc de Figure 3: Click EdU reaction in beta-actin-GFP expressing tissue Figure 3 : Optimizing the chelate concentration on beta-actin-GFP transgenic mouse duodenum. EdU treatment of 1 mg/20g body wt. i.p. 4 hour before sacrifice. Tissues were dissected, fixed 2 hours at RT in 4% PFA. They were then cryoprotected with a 30% sucrose solution and frozen in OCT. 8 m sections were labeled with unmodified Alexa Fluor ® 555 azide. Varying amounts of chelate were used to obtain optimal click and GFP signal. Low amounts of chelate preserves GFP fluorescence while giving detectable EdU signal. In 3a and 3b, even with higher exposure time, no EdU signal was detected. Figure 5: Modified azide dye enhances click signal Figure 7: Flow cytometry using U-2 OS cells transduced with Histone 2B-GFP and treated with EdU EdU Cu (I) DNA = Alexa Fluor ® dye 5-ethynyl-2’-deoxyuridine dual parameter plot GFP No chelate2X chelate2.6 X chelate Figure 6: Flow cytometry experiments using HCT116 Human fibroblasts transduced with lentiviral vector EGFP under control of promotor hPGK. Cells are treated with 20 M of EdU Figure 6 : Click-iT ® EdU Flow Cytometry assay. Comparison between different amounts of copper chelate. As expected, GFP fluorescence is conserved with lower amounts of chelate. No difference in GFP fluorescence is observed between unmodified and modified azide modified Alexa Fluor ® 647 dye. modified azidecopper chelate a+- b+1X c-2X a1 b1 b1b1 a2a2 b2b2 c2c2 2.4% 44% 50% c1c1 a1a1 DAPI EdU Count GFP DNA EdU a. b. Hoechst phalloidin GFP EU chelate amount GFP (10ms)EdU a4X+++- b3X+++- c2X ++++ d1X+++++ enone-+++ Figure 4 : Tissue prepared as described in fig. 3 was labeled with unmodified Alexa Fluor ® 555 azide using 2X chelate To assess the degree of labeling a second reaction was performed using unmodified Alexa Fluor ® 647 azide without chelate. All cells were double labeled, meaning that the chelate did not disturb the detection of the incorporated EdU. no copper Figure 5 : Tissue prepared as described in fig. 3 was labeled with modified or unmodified dye azide. 2X copper chelate was used to compare effect of modified azide on EdU and GFP signal. EdU signal from modified Alexa Fluor ® 594 azide click reaction in top row (a, c) is brighter than with unmodified azide click reaction in bottom row (b, d). e ca no chelate2X chelate b c d DAPI Alexa Fluor ® 647 signalAlexa Fluor ® 555 signal Overlay EdU 50ms EdU 500ms EdU 150msEdU 50ms abc de mod. azide chelate GFPEdU a+++++++ b-+++++ c+- - d---++ en/a +++- FxCycle™ Violet Click EdU GFP fluorescence N N N R′R′ R N N+N+ N-N- R′R′
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