Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

10.3791/60446 ◽  
2020 ◽  
Author(s):  
Grace A. Snyder ◽  
William E. Browne ◽  
Nikki Traylor-Knowles ◽  
Benyamin Rosental
Keyword(s):  
Cell Sorting ◽  
Cell Populations ◽  
Fluorescence Activated Cell Sorting

scholarly journals Fluorescence-Activated Cell Sorting Using the D-Root Device and Optimization for Scarce and/or Non-Accessible Root Cell Populations

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 499 ◽  
Author(s):  
Mary-Paz González-García ◽  
Estéfano Bustillo-Avendaño ◽  
Alvaro Sanchez-Corrionero ◽  
Juan C. del Pozo ◽  
Miguel A. Moreno-Risueno
Keyword(s):  
Cell Sorting ◽  
Fluorescent Protein ◽  
Rna Extraction ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Fluorescence Activated Cell Sorting ◽  
Stressful Environment ◽  
Minimum Number ◽  
Green Fluorescent

Fluorescence-activated cell sorting (FACS) is a technique used to isolate specific cell populations based on characteristics detected by flow cytometry. FACS has been broadly used in transcriptomic analyses of individual cell types during development or under different environmental conditions. Different protoplast extraction protocols are available for plant roots; however, they were designed for accessible cell populations, which normally were grown in the presence of light, a non-natural and stressful environment for roots. Here, we report a protocol using FACS to isolate root protoplasts from Arabidopsis green fluorescent protein (GFP)-marked lines using the minimum number of enzymes necessary for an optimal yield, and with the root system grown in darkness in the D-Root device. This device mimics natural conditions as the shoot grows in the presence of light while the roots grow in darkness. In addition, we optimized this protocol for specific patterns of scarce cell types inside more differentiated tissues using the mCherry fluorescent protein. We provide detailed experimental protocols for effective protoplasting, subsequent purification through FACS, and RNA extraction. Using this RNA, we generated cDNA and sequencing libraries, proving that our methods can be used for genome-wide transcriptomic analyses of any cell-type from roots grown in darkness.

scholarly journals A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

2021 ◽  
Vol 22 (6) ◽  
pp. 3041
Author(s):  
Gheorghita Menghiu ◽  
Vasile Ostafe ◽  
Radivoje Prodanović ◽  
Rainer Fischer ◽  
Raluca Ostafe
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Screening Method ◽  
Enzymatic Reaction ◽  
Hydrolytic Enzymes ◽  
Screening System ◽  
Fluorescence Activated Cell Sorting ◽  
Fungal Cell ◽  
Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

scholarly journals A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting

2021 ◽  
Vol 20 ◽  
pp. 587-600
Author(s):  
Elisa Murenu ◽  
Marina Pavlou ◽  
Lisa Richter ◽  
Kleopatra Rapti ◽  
Sabrina Just ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Bipolar Cells ◽  
Fluorescence Activated Cell Sorting ◽  
Universal Protocol
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