Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces.
We have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. This micro fluorescence-activated cell sorter (microFACS) uses an infrared laser to laterally deflect cells into a collection channel. Green-labeled macrophages were sorted from a 40/60 ratio mixture at a throughput of 22 cells/s over 30 min achieving a 93% sorting purity and a 60% recovery yield. To rule out potential photoinduced cell damage during optical deflection, we investigated the response of mouse macrophage to brief exposures (<4 ms) of focused 1064-nm laser light (9.6 W at the sample). We found no significant difference in viability, cell proliferation, activation state, and functionality between infrared-exposed and unexposed cells. Activation state was measured by the phosphorylation of ERK and nuclear translocation of NF-kappaB, while functionality was assessed in a similar manner, but after a lipopolysaccharide challenge. To demonstrate the selective nature of optical sorting, we isolated a subpopulation of macrophages highly infected with the fluorescently labeled pathogen Francisella tularensis subsp. novicida. A total of 10,738 infected cells were sorted at a throughput of 11 cells/s with 93% purity and 39% recovery.
Perroud
Thomas D
TD
Sandia National Laboratories, P.O. Box 969, Livermore, California 94551, USA.
Kaiser
Julia N
JN
Sy
Jay C
JC
Lane
Todd W
TW
Branda
Catherine S
CS
Singh
Anup K
AK
Patel
Kamlesh D
KD
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D003952:Diagnostic Imaging; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes; D005604:Francisella tularensis / Q000276:immunology / Q000378:metabolism / Q000528:radiation effects*; D049452:Green Fluorescent Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000528:radiation effects; D008264:Macrophages / Q000276:immunology / Q000382:microbiology / Q000528:radiation effects*; D051379:Mice; D044085:Microfluidics / Q000379:methods*; D016328:NF-kappa B / Q000378:metabolism; D052898:Optical Tweezers; D010766:Phosphorylation / Q000528:radiation effects; D021381:Protein Transport; D015398:Signal Transduction; D014406:Tularemia / Q000276:immunology
D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D016328:NF-kappa B; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D048049:Extracellular Signal-Regulated MAP Kinases