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
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hydrochloric acid, ACS reagent, 37%
Sigma-Aldrich
Penicillin-Streptomycin, Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Fetal Bovine Serum, USA origin, sterile-filtered, suitable for cell culture, suitable for hybridoma
Sigma-Aldrich
D-(+)-Glucose, ≥99.5% (GC)
Sigma-Aldrich
L-Glutamine solution, 200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
Sigma-Aldrich
Trypsin-EDTA solution, 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
Sigma-Aldrich
Sucrose, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Sodium hydroxide, reagent grade, ≥98%, pellets (anhydrous)
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Ammonium persulfate, for molecular biology, suitable for electrophoresis, ≥98%
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Ethylenediaminetetraacetic acid disodium salt dihydrate, suitable for electrophoresis, for molecular biology, 99.0-101.0% (titration)
Sigma-Aldrich
N,N,N′,N′-Tetramethylethylenediamine, BioReagent, suitable for electrophoresis, ~99%
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Trizma® base, BioPerformance Certified, meets EP, USP testing specifications, suitable for cell culture, ≥99.9% (titration)
Sigma-Aldrich
Trizma® hydrochloride, reagent grade, ≥99.0% (titration), crystalline
Sigma-Aldrich
MOPS, ≥99.5% (titration)
SAFC
Phosphate Buffered Saline, pH 7.2, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Calcium chloride dihydrate, ACS reagent, ≥99%
Sigma-Aldrich
Citric acid, 99%
Sigma-Aldrich
Hanks′ Balanced Salt solution, Modified, with sodium bicarbonate, without phenol red, liquid, sterile-filtered, suitable for cell culture
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Sodium acetate, anhydrous, ReagentPlus®, ≥99.0%
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Potassium phosphate monobasic, ReagentPlus®
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Trisodium citrate dihydrate, meets USP testing specifications
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Sodium phosphate dibasic, BioXtra, ≥99.0%
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Acrylamide/Bis-acrylamide, 30% solution, BioReagent, suitable for electrophoresis, 37.5:1
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Dulbecco′s Modified Eagle′s Medium - high glucose, 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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Sodium tetraborate decahydrate, ReagentPlus®, ≥99.5%
Sigma-Aldrich
Magnesium sulfate, BioReagent, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
Boric acid, ReagentPlus®, ≥99.5%
Sigma-Aldrich
Poly-L-lysine hydrobromide, mol wt ≥300,000
Sigma-Aldrich
Deoxyribonucleic acid, single stranded from salmon testes, For hybridization