Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal

10.3791/62883 ◽  
2021 ◽  
Author(s):  
Quentin Amossé ◽  
Kelly Ceyzériat ◽  
Stergios Tsartsalis ◽  
Benjamin B. Tournier ◽  
Philippe Millet
Keyword(s):  
Cell Sorting ◽  
Fluorescence Activated Cell Sorting ◽  
Cellular Origin

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

Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies

2017 ◽  
Vol 89 (7) ◽  
pp. 4007-4012 ◽  
Author(s):  
Martin Dippong ◽  
Peter Carl ◽  
Christine Lenz ◽  
Jörg A. Schenk ◽  
Katrin Hoffmann ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Single Cell ◽  
Cell Sorting ◽  
Cell Selection ◽  
Fluorescence Activated Cell Sorting ◽  
Selection Of

Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures

2010 ◽  
Vol 5 (12) ◽  
pp. 1993-2004 ◽  
Author(s):  
Günter Mayer ◽  
Marie-Sophie L Ahmed ◽  
Andreas Dolf ◽  
Elmar Endl ◽  
Percy A Knolle ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Fluorescence Activated Cell Sorting
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