Microfluidic-Based Cell Sorting of Francisella tularensis Infected Macrophages Using Optical Forces

• Published in: Analytical chemistry (Washington) Vol. 80; no. 16; pp. 6365 - 6372
• Main Authors: Perroud, Thomas D, Kaiser, Julia N, Sy, Jay C, Lane, Todd W, Branda, Catherine S, Singh, Anup K, Patel, Kamlesh D
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.08.2008
• Subjects: Analytical chemistry; Animals; Bacteria; Cell Nucleus - metabolism; Cell Proliferation; Cell Separation - methods; Cell Survival; Cells; Cells, Cultured; Chemistry; Diagnostic Imaging; Exact sciences and technology; Extracellular Signal-Regulated MAP Kinases - metabolism; Flow Cytometry; Fluorescence; Fluorescent Dyes; Francisella tularensis; Francisella tularensis - immunology; Francisella tularensis - metabolism; Francisella tularensis - radiation effects; Green Fluorescent Proteins; Infrared radiation; Lipopolysaccharides - pharmacology; Macrophage Activation - radiation effects; Macrophages - immunology; Macrophages - microbiology; Macrophages - radiation effects; Mice; Microfluidics - methods; NF-kappa B - metabolism; Optical Tweezers; Optics; Phosphorylation - radiation effects; Protein Transport; Signal Transduction; Spectrometric and optical methods; Tularemia - immunology; Fluid mechanics; Cell culture; Flow cytometry; Cell sorter; Purity; Bacteria; Confocal microscopy; Optical forces; Microfluidics; Fluorescence spectrometry; Francisella tularensis; Macrophage
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac8007779

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 (µFACS) 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-κB, 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.