Release of intracellular proteins by electroporation with preserved cell viability.

Extraction of intracellular proteins from cells is often an important first step for conducting molecular biology and proteomics studies. Although ultrasensitive detection and analytical technology at the single molecule level is becoming routine, protein extraction techniques have not followed suit and still call for complete lysis that leads to cell death. In principle, with refined extraction techniques, intracellular proteins can potentially be extracted without killing the cell. In this Letter, we demonstrate that electroporation is capable of releasing intracellular proteins from adherent Chinese hamster ovary cells while preserving the cell viability. By tuning the duration and intensity of an electric pulse, we were able to control the average amount of protein release and the percentage of viable cells after the operation. Our results indicate that a substantial fraction of the cell population was able to release proteins under electroporation and survive the procedure. Interestingly, at the single cell level, the probability for cell death does not increase with more protein release. This work paves the way to extracting and analyzing intracellular proteins while keeping cells live.

[1]  Chang Lu,et al.  Microfluidic electroporation of tumor and blood cells: observation of nucleus expansion and implications on selective analysis and purging of circulating tumor cells. , 2010, Integrative biology : quantitative biosciences from nano to macro.

[2]  Bo Huang,et al.  Counting Low-Copy Number Proteins in a Single Cell , 2007, Science.

[3]  D. Baltimore,et al.  Achieving Stability of Lipopolysaccharide-Induced NF-κB Activation , 2005, Science.

[4]  Timothy K Lee,et al.  Single-cell NF-κB dynamics reveal digital activation and analogue information processing , 2010, Nature.

[5]  J E Ferrell,et al.  The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes. , 1998, Science.

[6]  M. McClain,et al.  Microfluidic devices for the high-throughput chemical analysis of cells. , 2003, Analytical chemistry.

[7]  N. Anderson,et al.  Proteome and proteomics: New technologies, new concepts, and new words , 1998, Electrophoresis.

[8]  Uri Alon,et al.  Dynamics of the p53-Mdm2 feedback loop in individual cells , 2004, Nature Genetics.

[9]  Yiqiong Zhao,et al.  Using polarization-shaped optical vortex traps for single-cell nanosurgery. , 2007, Nano letters.

[10]  N. Allbritton,et al.  Micropatterning of living cells on a heterogeneously wetted surface. , 2006, Langmuir : the ACS journal of surfaces and colloids.

[11]  Chang Lu,et al.  Microfluidic chemical cytometry based on modulation of local field strength. , 2006, Chemical communications.

[12]  T. Geng,et al.  Observing single cell NF-κB dynamics under stimulant concentration gradient. , 2012, Analytical chemistry.

[13]  K. Jensen,et al.  A microfluidic electroporation device for cell lysis. , 2005, Lab on a chip.

[14]  Tomoyuki Iwasawa,et al.  Single-cell chemical lysis method for analyses of intracellular molecules using an array of picoliter-scale microwells. , 2008, Analytical chemistry.

[15]  Chang Lu,et al.  One-step extraction of subcellular proteins from eukaryotic cells. , 2010, Lab on a chip.

[16]  Chang Lu,et al.  High‐throughput and real‐time study of single cell electroporation using microfluidics: Effects of medium osmolarity , 2006, Biotechnology and bioengineering.

[17]  Rong Fan,et al.  Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells , 2011, Proceedings of the National Academy of Sciences.

[18]  Mark Bachman,et al.  Fast-lysis cell traps for chemical cytometry. , 2008, Lab on a chip.

[19]  D. Chiu,et al.  Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets. , 2005, Analytical chemistry.

[20]  M J May,et al.  NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. , 1998, Annual review of immunology.

[21]  A. Hoffmann,et al.  Circuitry of nuclear factor κB signaling , 2006 .

[22]  Chang Lu,et al.  Microfluidic electroporation for selective release of intracellular molecules at the single‐cell level , 2008, Electrophoresis.

[23]  N. Allbritton,et al.  Spatial control of cellular measurements with the laser micropipet. , 2001, Analytical chemistry.

[24]  Vasan Venugopalan,et al.  Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging. , 2008, Lab on a chip.

[25]  R. Rosenfeld Nature , 2009, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.

[26]  B. Tromberg,et al.  Characterization of cellular optoporation with distance. , 2000, Analytical chemistry.

[27]  Paul C. H. Li,et al.  Transport, manipulation, and reaction of biological cells on-chip using electrokinetic effects. , 1997, Analytical chemistry.

[28]  James E. Ferrell,et al.  Bistability in cell signaling: How to make continuous processes discontinuous, and reversible processes irreversible. , 2001, Chaos.

[29]  Y. Zhan,et al.  Kinetics of NF-κB nucleocytoplasmic transport probed by single-cell screening without imaging. , 2010, Lab on a chip.

[30]  Lidong Qin,et al.  Self-powered microfluidic chips for multiplexed protein assays from whole blood. , 2009, Lab on a chip.