Simultaneous quantitative determination of electroporative molecular uptake and subsequent cell survival using gel microdrops and flow cytometry.
BACKGROUND
Electroporation is widely used to introduce molecules into cells, but conditions yielding maximal molecular uptake often result in low cell survival. We describe a high throughput method for analyzing populations of culturable cells simultaneously for molecular uptake and cell growth.


METHODS
Cells are microencapsulated within agarose gel microdrops (GMDs), exposed to a polar tracer fluorescent molecule, electrically pulsed at various field strengths, and cultured. The GMDs are then analyzed at about 100,000 occupied GMDs per hour by flow cytometry for both uptake and microcolony formation.


RESULTS
We demonstrate how the method can be used to optimize a parameter of interest (e.g., the applied field strength) with respect to both uptake and cell survival. Here, the optimal field strength is determined to be 1.7 kV/cm. Below this, there is lower molecular uptake. As the field strength is increased, the cell survival rate goes down.


CONCLUSIONS
This method may be applicable to optimization of other electroporation parameters and alternative physical and chemical methods for cell loading.
Gift
E A
EA
Harvard-M.I.T. Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Weaver
J C
JC
D000818:Animals; D002214:Capsules; D015169:Colony Count, Microbial / Q000379:methods; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D018274:Electroporation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005782:Gels; D007202:Indicators and Reagents; D051379:Mice; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000254:growth & development*; D014162:Transfection / Q000379:methods
D002214:Capsules; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005782:Gels; D007202:Indicators and Reagents; C007740:fluorexon