Optimization of reporter cells for expression profiling in a microfluidic device.
The emergence of green fluorescence protein (GFP) technologies has enabled non-invasive monitoring of cell function and gene expression. GFP-based expression studies are typically performed in traditional single-dish or multi-well formats to monitor a small number of genes or conditions that do not lend well to scaling, high-throughput analysis, or single-cell measurements. We have recently developed a microfluidic device, the Living Cell Array (LCA), for monitoring GFP-based gene expression in a high-throughput manner. Here, we report the optimization of GFP reporter cell characteristics in this microfluidic device for gene expression profiling. A reporter cell line for the transcription factor NF-kappa B was generated and used as the model cell line. Reporter cells were seeded in the LCA and NF-kappa B activated by addition of the cytokine TNF-alpha . Our studies show that the fluorescence kinetics from the reporter cell line in response to both single and repeated TNF-alpha stimulation in the LCA is similar to that observed in standard tissue culture. In addition, our data also indicate that multiple expression waves can be reliably monitored from a small population of reporter cells. Using reporter cell line subcloning and cell cycle synchronization, we demonstrate that the kinetics and magnitude of induced fluorescence in the reporter cell lines can be further improved to maximize the fluorescence readout from reporter cell lines, thereby improving their applicability to live cell expression profiling. Our studies establish some of the important criteria to be considered when using reporter cell lines for dynamic expression profiling in microfluidic devices.
Wieder
Kenneth J
KJ
Center for Engineering in Medicine/Department of Surgery, Massachusetts General Hospital, Shriners Hospitals for Children, Harvard Medical School, Boston, MA 02114, USA.
King
Kevin R
KR
Thompson
Deanna M
DM
Zia
Cindy
C
Yarmush
Martin L
ML
Jayaraman
Arul
A
D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods; D020869:Gene Expression Profiling / Q000295:instrumentation* / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D016328:NF-kappa B / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism*
D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins