Single Cell Analysis and Sorting of Aspergillus fumigatus by Flow Cytometry
用流式细胞仪对烟曲霉进行单细胞分析和分选
发布: 2021年04月20日第11卷第8期 DOI: 10.21769/BioProtoc.3993 浏览次数: 4348
评审: Emilia KrypotouPreeti SharmaSimab Kanwal
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参见作者原研究论文
The authors used this protocol in:
May 2020
Abstract
Experimental results in fungal biology research are usually obtained as average measurements across whole populations of cells, whilst ignoring what is happening at the single cell level. Microscopy has allowed us to study single-cell behavior, but it has low throughput and cannot be used to select individual cells for downstream experiments. Here we present a method that allows for the analysis and selection of single fungal cells in high throughput by flow cytometry and fluorescence activated cell sorting (FACS), respectively. This protocol can be adapted for every fungal species that produces cells of up to 70 microns in diameter. After initial setting of the flow cytometry gates, which takes a single day, accurate single cell analysis and sorting can be performed. This method yields a throughput of thousands of cells per second. Selected cells can be subjected to downstream experiments to study single-cell behavior.
Keywords: Flow cytometry (流式细胞术)
FACS (流式细胞荧光分选技术)
Fungi (真菌)
Spore (孢子)
Germination (发芽)
Fluorescence (荧光)
Single cell (单细胞)
High throughput (高通量)
Background
Fungal biology research has often been dependent on average measurements of total populations of cells, thereby missing what is happening at the individual cell level. This is specifically the case for filamentous fungi, since hyphae grow by apical extension and branch subapically, and may fuse with neighboring hyphae. This behavior results in an interconnected, entangled mass of hyphae, the colony, which makes it challenging to study single-cell behavior. Therefore, most research on single cells has focused on non-overlapping hyphae that are located in the colony margin using microscopy (Vinck et al., 2005 and 2011; Bleichrodt et al., 2012 and 2015). Since hyphae are attached to the colony, it is challenging to segment them using image analysis. Here we developed a flow cytometry protocol to analyze and select single cells with high throughput. This protocol is suited for e.g. analyzing germination of spores; selection of fluorescent transformants; minimum inhibitory concentration (MIC) testing; obtaining cells for single cell omics approaches; identifying heterogeneous subpopulations of cells; cell aggregation assays, which can be relevant for studying the initial stages of pellet formation in fermenters; and analysis and selection of any fluorescently labeled cellular target, whether stained or genetically encoded, that is smaller than 70 microns (Bleichrodt and Read, 2019).
Materials and Reagents
Falcon 5 ml flow cytometry tubes (STEMCELL Technologies, catalog number: 38057)
Corning cell culture flasks, 25 cm2, vented cap (Merck, catalog number: CLS430639-200EA)
Inoculation loops (VWR, catalog number: SIMPL200-2)
Sterile cotton swabs (VWR, catalog number: HERE1030619)
Easystrainer 70 µm filter, for 50 ml tubes (Greiner Bio-One, catalog number: 542070)
Corning 50 ml centrifuge tubes (Merck, catalog number: CLS430828-100EA)
1.5 ml Eppendorf tubes (VWR International, catalog number: 0030125150)
Microscopy slides and coverslips
96-well plates, optically clear bottom µ-Plate, 96 Well Black (Ibidi, catalog number: 89626)
Pipette filter tips
10 ml pipettes (VWR, catalog number: BURK7550-0010)
4-peak beads (Spherotech, Lake Forest, IL, catalog number: RCP-35-5). Store at 4 °C for up to 1 year
Accudrop beads (Beckton Dickinson Biosciences, catalog number: 345249). Store at 4 °C for up to 1 year
Calcium-/magnesium-free phosphate buffered saline (PBS) tablets (ThermoFisher Scientific, Gibco, catalog number: 18912014)
Ethanol (Fisher Scientific, catalog number: 12468750) diluted to 70% v/v with Milli-Q water. Store at room temperature (RT) for up to 1 year
Sodium hypochlorite (Fisher Scientific, catalog number: 11448842). Store at room temperature for up to 1 year
Potato dextrose agar (Merck Millipore, catalog number: 1.10130.0500)
Fluorescent Brightener 28 disodium salt solution (Calcofluor white solution), 25% (Merck, catalog number: 910090-20ML, CAS Number: 4193-55-9). Store at 4 °C for up to 1 year
Sheath fluid (see Recipes)
50% glucose solution (see Recipes)
Minimal medium (MM) (see Recipes)
Phosphate-buffered saline (see Recipes)
Saline Tween solution (ST) (see Recipes)
Calcofluor white working solution (see Recipes)
Vishniac solution (see Recipes)
Equipment
Hemocytometer (VWR, catalog number: BRND718905)
Set of micropipettes
BD Influx cell sorter (Becton Dickinson) with 100-µm nozzle tip
Sonicating water bath (Grant Instruments Ltd, model XUBA3)
Autoclave (Prestige Electrolysis + Spa Supply, model: MED 2100, catalog number: SAP2100)
Temperature-controlled incubators (Binder, Series BD Classic Line, model: BD 400)
Light microscope (ZEISS, model: Axiolab 5)
Fluorescence microscope (ZEISS, Axio Observer Research Inverted Microscope)
Pipet controller (VWR, model: Pipet boy, catalog number: 612-0928)
Tabletop centrifuge (Fisher Scientific, model: EppendorfTM Benchtop Microcentrifuge, catalog number: 10216522)
Software
Sortware version 1.2.0.142 (Becton Dickinson)
FlowJo version 10.7 (Becton Dickinson) (https://www.flowjo.com/)
Rstudio version 1.3 (optional) (https://rstudio.com/products/rstudio/)
Procedure
文章信息
版权信息
© 2021 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Howell, G. J. and Bleichrodt, R. (2021). Single Cell Analysis and Sorting of Aspergillus fumigatus by Flow Cytometry. Bio-protocol 11(8): e3993. DOI: 10.21769/BioProtoc.3993.
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分类
微生物学 > 微生物细胞生物学 > 基于细胞的分析方法
细胞生物学 > 单细胞分析 > 流式细胞术
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