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Merck

Versatile phenotype-activated cell sorting.

Science advances (2020-10-25)
Jihwan Lee, Zhuohe Liu, Peter H Suzuki, John F Ahrens, Shujuan Lai, Xiaoyu Lu, Sihui Guan, François St-Pierre
RESUMO

Unraveling the genetic and epigenetic determinants of phenotypes is critical for understanding and re-engineering biology and would benefit from improved methods to separate cells based on phenotypes. Here, we report SPOTlight, a versatile high-throughput technique to isolate individual yeast or human cells with unique spatiotemporal profiles from heterogeneous populations. SPOTlight relies on imaging visual phenotypes by microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. To illustrate SPOTlight's ability to screen cells based on temporal properties, we chose to develop a photostable yellow fluorescent protein for extended imaging experiments. We screened 3 million cells expressing mutagenesis libraries and identified a bright new variant, mGold, that is the most photostable yellow fluorescent protein reported to date. We anticipate that the versatility of SPOTlight will facilitate its deployment to decipher the rules of life, understand diseases, and engineer new molecules and cells.

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Ácido clorídrico, ACS reagent, 37%
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Hidróxido de sódio, reagent grade, ≥98%, pellets (anhydrous)
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Penicilina-estreptomicina, Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
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Soro bovino fetal, USA origin, sterile-filtered, suitable for cell culture, suitable for hybridoma
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Sacarose, for molecular biology, ≥99.5% (GC)
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D-(+)-Glicose, ≥99.5% (GC)
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Trizma® base, BioPerformance Certified, meets EP, USP testing specifications, suitable for cell culture, ≥99.9% (titration)
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Acetato de sódio, anhydrous, ReagentPlus®, ≥99.0%
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Cloreto de cálcio, ACS reagent, ≥99%
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Solução de tripsina-EDTA, 1 ×, sterile; sterile-filtered, BioReagent, suitable for cell culture, 0.5 g porcine trypsin and 0.2 g EDTA, 4Na per liter of Hanks′ Balanced Salt Solution with phenol red
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Ácido etilenodiamino tetra-acético, suitable for electrophoresis, for molecular biology, 99.0-101.0% (titration)
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Cloridrato de Trizma®, reagent grade, ≥99.0% (titration), crystalline
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L-Glutamina, 200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
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MOPS, ≥99.5% (titration)
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Cloreto de sódio, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
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Persulfato de amônio, for molecular biology, suitable for electrophoresis, ≥98%
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N,N,N′,N′-tetrametiletilenodiamina, BioReagent, suitable for electrophoresis, ≥99.0%
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Solução salina balanceada de Hank, Modified, with sodium bicarbonate, without phenol red, liquid, sterile-filtered, suitable for cell culture
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Ácido bórico, ReagentPlus®, ≥99.5%
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Sodium tetraborate decahydrate, ReagentPlus®, ≥99.5%
SAFC
Solução salina tamponada com fosfato, pH 7.2, liquid, sterile-filtered, suitable for cell culture
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Acrilamida/Bisacrilamida, 30%, BioReagent, suitable for electrophoresis, 37.5:1
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Sulfato de magnésio, BioReagent, suitable for cell culture, suitable for insect cell culture
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Meio de Eagle modificado por Dulbecco - alto teor de glicose, With 4500 mg/L glucose and sodium bicarbonate, without L-glutamine, sodium pyruvate, and phenol red, liquid, sterile-filtered, suitable for cell culture
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Deoxyribonucleic acid, single stranded from salmon testes, For hybridization
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Poli-L-lisina, mol wt ≥300,000
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Ácido cítrico, 99%
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Fosfato de potássio, ReagentPlus®
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Citrato de sódio tribásico, meets USP testing specifications
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2-Phenylindole, technical grade, 95%