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Imaging for Degradation of IKB-EGFP in a Single Jurkat T Cell Studied Within a Microfluidic Channel

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Imaging for Degradation of IKB-EGFP in a Single Jurkat T Cell Studied Within a Microfluidic Channel Imaging for Degradation of IKB-EGFP in a single Jurkat T Cell studied within a Microfluidic Channel Paul C. H. Li,* Laurent de Camprieu and Monika Sangar Department of Chemistry, Simcm Fraser University, 8888 University Drive, Burnaby, BC, Canada, V5A 12% *paulZi~fu. ca Abstract For the first time, we report a real-time cellular change on a single Jukat T cell residing inside a microfluidic device consisting of a microchmel. Fluorescent imaging on the same, single suspension cellwas carried out to study a cellular process occurring in viable cells: degradation of an inhibitory protein, It&, which is involved in the NF-KB signaling pathway. The decrease in the intensity of EGFP due to the degradation of the IKB-EGFP fusion protein was monitored. The correlation between the change in fluorescent intensity and cell parameters (i.e. cell size, environments) was discussed. It is demonstrated that the microfluidic chip can facilitate monitoring of cellular changes in the single cell level and for non-adherent cells. Keywords: non-adherent single cells, fluorescent imaging, IKB protein, TNF-a 1. Introduction The impact of microfluidics on modern cellular and molecular biology methodologies, such as cell manipulation,’ green fluorescent protein (GFP) expression2 gene transfection3 and cellular calcium flux4 has been demonstrated. However, no work has been carried out to study more sophisticated biochemical systems on-chip. As reported previously, degradation of IKB leads to the activation of the transcription factor: NF-KB.~~ 6 The NF-KB pathway has been involved in pathogenesis of autoimmune diseases, inflammatory diseases, tumor growth and metastasis.7. 8 Therefore, this work has strong implication in the use of the NF-KB pathway to screen anti-tumor drugs. 2. Experimental Cell culture and transfection were performed in a biosafety cabinet Class II Type A/B3 (NuAire). Incubation of cells for cell growth and gene expression was conducted in an air-jacketed DH Autoflow CO2 incubator (NuAire). Transient transfection of a reported vector (~IKB-EGFP) to Jurkat T cells was achieved by chemical and electroporation methods. The fluorescence imaging system (Vanox AHBS3) consists of an upright microscope (Olympus AH-3) and a CCD color video camera (Sony). The etched microfluidic glass plates, which consisted of several microchannels, were fabricated at Alberta Microelectronic Centre (now Micralyne). The cover plate was perforated with holes for liquid access using a mechanical drill. Both the channel and 7th lnternat~onal Conference on Miniaturized Chemical and Blochemlcal Analysts Systems October 5-9, 2003, Squaw Valley, Callfornla USA O-974361 I-0.O/~TAS2003/$15.0002003TRF 1149 cover plates were cleaned with detergent and distilled water inside an in-house built microchip washer. This apparatus eliminates the further need of clean room facilities after the channel plates were fabricated. The relevant portion of the finished chip used in the experiments was depicted in Figure la. In most cases, the straight channel, which was about 2 cm long, 1830 pm wide and 15 CLm deep, was employed. The channel served as a reagent delivery path Figure 1. (a) A ~nic~“~~~~dicchip (ofIO cm side) showing and observation zone the relevant channels (430 and 1830 pm wide) for for cells, media and jluorescent imaging experiments, (b) A few Jurknt cells reagents. Numerous (about 10 pm in diameter) introduced into a microchannel Jurkat cells that were of15 pm deep as observed using an upright microscope introduced in the with a 20X objective. Note the channel wall on the left side microchannel are ofthe image. shown in Figure lb. By liquid pressure difference, transfected Jurkat T cells were first into a microchannel of a microchip (see Figure la). The cells were able to remain stationary by adhering to the bottom of the microchannel (see Figure lb). Then a cytokine: TNF-a (10 ng/mL) was introduced to stimulate the transfected cells which was fluorescent. The decrease in the fluorescent intensity of enhanced green fluorescent protein (EGFP) due to the proteolytic degradation of It&-EGFP in the one transfected cell was monitored. Images were taken at 7 time points (i.e. 0, 5, 10, 15, 20, 25, 30 min.). The results gathered from fluorescent experiments were used to determine the half-lives of IKB-EGFP degradation in Jurkat T cell. 3. Results and Discussion Figure 2b-h show the fluorescent images (negative) of the cell after treatment of TNF-a over a period of 30 min. Stimulation by TNF-a caused the fluorescence intensity 7th lnternat~onal Conference on Miniaturized Chemical and Blochemlcal Analysts Systems October 5-9, 2003, Squaw Valley, Callfornla USA 1150 Figure 2. (a) The brightfield image of a transfected Jwkat T-cell by electroporation plus a non-transfected cell, and the fluorescent images (negative) of the II&-EGFP- expressing cell at (b) 0 min, (c) 5 min, (d) 10 min, (e) 15 min, (f) 20 min, (g) 25 min, (h) 30 min. after TNF- a (10 ng/mL) treatment. The scale bars in all images represent 10 pm. These data correspond to experiment 13 (transfection by electroporation). decreases. As shown in the brightfield image (Figure 2a), this transfected cell is found to be adjacent to a non-transfected cell. In addition, the two cells are beside a large cellular debris. The presence of an adjacent fluorescent entity (presumably from cellular debris) in the field of view provided a negative control. The mean fluorescent intensity from the same cell at each time point was determined according to a constant image area. After normalization, the relative fluorescent intensity was plotted as a function of time for this imaging experiment (13) for an electroporated cell and other experiments, as shown in Figure 3. The percentage fluorescence decrease for this experiment (13) was determined to be 58%. Moreover, the half-life was estimated to be 21 min. As for the chemically transfected cell (experiment 5), the percentage fluorescence decrease was determined to be 67%, and the half-life was 25 min. The decrease in fluorescent intensity plus increase in degradation half-lives were found to be greater in stimulated cells versus control cells transfected by both methods, see Fig. 3. However, the decrease in controls was found to be greater in electroporated cells than in chemically transfected cells, possibly because of EGFP leakage through the cell membrane. With the bright field and fluorescent images available, correlation between the fluorescent intensity changes with cell size, IKB-EGFP expression level and cell neighborhood can be inferred. No fluorescent imaging has ever been carried out on the TNF-a stimulation on non-adherent Jurkat cells. The close ones are Western blotting data of It& degradation of batches of Jurkat cells due to ‘INF-a9. lo or fluorescent imaging data of. IKE&EGFP degradation in single HeLa cells due to IL- 1p.r’ Future work include the testing of various therapeutic reagents on the degradation of the fusion protein. 7th lnternat~onal Conference on M~n~atur~zed Chemical and Blochemlcal Analysts Systems October 5-9, 2003, Squaw Valley, Callfornla USA 1151 Acknowledgements We are grateful to Canada Foundation for Innovation, Natural Sciences and Research Engineering Council of Canada and British Columbia Knowledge Development Fund for financial support. LC is grateful to SFU for a graduate scholarship. We (”“..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “..” “. acknowledge p i invaluable assistance from expt 12 Rosemary Cornell expt la and Ming Tang Xie for sharing expt 13 their molecular expt 5 biology expertise and facilities, YJ ; Ingrid Northwood / and Teresa Kittos 0 5 10 15 20 25 30 for cell culture Time (min) expertise. We are also indebted to VWRCanlab for Figure 3. Change of relative fluorescent intensity (after the loan and normalization to give 100% at 0 min) of Jurkat cells expressing evaluation of an It@-EGFP as a function of time for TNF- a experiments 5 and electroporator for 13 and for control experiments 12 and 18. Experiments 5 and transfection 12 were performed on chemically transfected cells, and experiments. experiments 13 and I8 on electroporated cells. References [l] Li, P.C.H. andD.J. Harrison. 1997. Anal. Chem. 69:1564-8. [2] Fu, A.Y., C. Spence, A. Scherer, F.H. Arnold and S.R. Quake. 1999. Nat. Biotechnol, 17: 1109-l 1. [3] Lin, Y.C., C.M. Jen, M.Y. Huang, C.Y. Wu and X.Z. Lin. 2001. Sens. Actuators B 79:137-43. [4] Yang, M., Li, C.W. and Yang, J. 2002. Anal. Chem. 74:3991-4001. [S] Davis, N., S. Ghosh, D.L. Simmons, P. Tempst, H.C. Lion, D. Baltimore and H.R. Bose. 1991. Science 253:1268-71. [6] Baeuerle, P.A. and D. Baltimore. 1988. Cell 53:211-17. [7] Nakshatri, H., P. Bhat-Nakshatri, D.A. Martin, R.J. Goulet Jr. and G.W. Sledge Jr. 1997. Mol. Cell. Biol. 17:3629-39. [8] Schwartzman, R.A. and J.A. Cidlowski. 1993. Endocr. Rev. 14:133-51. [9] Majumdar, S. and B.B. Aggarwal. 2001. J. Immunol. 167:2911-20. [lo] Sun, SC., P.A. Ganchi, D.W. Ballard and W.C. Greene. 1993. Science 259:1912-15. [ll] Yang, L., Chen, H. and E. Qwarnstrom.. Biochem. Biophys. Res. Commun. 2001, 285:603-O&. 7th lnternat~onal Conference on Miniaturized Chemical and Blochemlcal Analysts Systems October 5-9, 2003, Squaw Valley, Callfornla USA 1152 .
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