MicroPrep: chip-based dielectrophoretic purification of mitochondria.
We have developed a microfluidic system--microPrep--for subcellular fractionation of cell homogenates based on dielectrophoretic sorting. Separation of mitochondria isolated from a human lymphoblastoid cell line was monitored by fluorescence microscopy and further characterized by western blot analysis. Robust high throughput and continuous long-term operation for up to 60 h of the microPrep chip system with complex biological samples became feasible as a result of a comprehensive set of technical measures: (i) coating of the inner surfaces of the chip with BSA, (ii) application of mechanical actuators to induce periodic flow patterns, (iii) efficient cooling of the device to ensure integrity of organelle, (iv) a wide channel to provide for high fluidic throughput, and (v) integration of a serial arrangement of 10 dielectrophoretic deflector units to enable separation of samples with a high particle load without clogging. Hence, microPrep yields tens of micrograms of enriched and purified mitochondria within hours. Western blots of mitochondria fractions showed that contaminating endoplasmatic reticulum was reduced by a factor 6 when compared with samples prepared by state of the art centrifugation.
Moschallski
Meike
M
Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany.
Hausmann
Monika
M
Posch
Anton
A
Paulus
Aran
A
Kunz
Nancy
N
Duong
Thanh Tu
TT
Angres
Brigitte
B
Fuchsberger
Kai
K
Steuer
Heiko
H
Stoll
Dieter
D
Werner
Simon
S
Hagmeyer
Britta
B
Stelzle
Martin
M
D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D045744:Cell Line, Tumor; D046209:Electrophoresis, Microchip / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D006801:Humans; D008928:Mitochondria / Q000737:chemistry*; D024101:Mitochondrial Proteins / Q000032:analysis*
D024101:Mitochondrial Proteins