Flow cytometry: retrospective, fundamentals and recent instrumentation

Picot, Julien; Guerin, Coralie L.; Kim, Caroline Le Van; Boulanger, Chantal M.

doi:10.1007/s10616-011-9415-0

Cited by 201 publications

(175 citation statements)

References 60 publications

(47 reference statements)

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“…2,000 μm (31). The network structure of the pellets appears as a mixture of multiple layers of cells on a microscope slide, with the sizes of the pellets being too large to pass through the nozzle of a commercial FACS machine (32). This led to no description of the GFP-based quantitative measurement in Streptomyces.…”

Section: Significancementioning

confidence: 99%

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

Bai

Zhang

Zhao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

168

197

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There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.synthetic biology | natural product | flow cytometry | single-cell resolution | modular regulatory elements S treptomycetes are well known as the most abundant source of bioactive secondary metabolites (1), including medically important antimicrobial agents [e.g., chloramphenicol from Streptomyces venezuelae (2)], agricultural chemicals [e.g., avermectin from Streptomyces avermitilis (3)], and anticancer agents and immunosuppressants [e.g., rapamycin from Streptomyces hygroscopicus (4)]. However, the increasing difficulty of discovering novel drugs via traditional high-throughput screening and the "one strain many compounds" approach is frustrating pharmaceutical productivity (5, 6). Deciphering the genome sequences of Streptomyces surprisingly established the presence of a plethora of gene clusters encoding for yet-unobserved molecules, even in intensively investigated Streptomyces coelicolor A3 (2), revealing a much higher potential of novel bioactive agent production than originally anticipated (7,8). Therefore, the enormous number of natural products that have been obtained likely represent only a tiny portion of the repertoire of bioactive compounds that can possibly be produced. This has brought about extensive research into applied genomics aimed at investigating these new gene clusters, generally referred to as "cryptic," "silent," or "orphan" (9-11). With data on more than 12,000 in-house draft bacterial genomes, the potential for the discovery of a number of novel chemicals encrypted in silent biosynthetic gene clusters has been detected by genome mining.Many new strategies have been documented for "awakening" poorly expressed and/or silent gene clusters in Strept...

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Section: Significancementioning

confidence: 99%

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

Bai

Zhang

Zhao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

168

197

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“…Blood samples (5-10 μL of each fish) were first treated with DAPI DNA staining solution for 10-15 min, and then filtered. The DNA content was quantified by flow cytometer (Beckman Coulter Cell Lab QuantaTM SC) as described (Wei et al, 2003;Wang et al, 2011;Picot et al, 2012). A total of 450 tetraploid individuals were detected, and 10 gibel carp clone D individuals with 162 chromosomes (Wang et al, 2011), 10 domestic goldfish with 100 chromosomes (Song et al, 2012), and 10 individuals of the improved red common carp variety with 100 chromosomes (Jiang et al, 1983;Zhou et al, 1999) were also checked to set as the controls.…”

Section: Dna Content Measurement and Chromosome Number Countingmentioning

confidence: 99%

A novel allotetraploid gibel carp strain with maternal body type and growth superiority

Liang

Wang

et al. 2016

Aquaculture

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“…Flow cytometry is a fast and versatile technique that allows the detailed phenotypic characterization of the cells, allowing the study of cell maturation, activation and apoptosis related markers in simultaneously, as well as functional studies [26]. The identification and characterization of EC by FCM must be carried out carefully, concerning not only the method used to isolate the EC, but also the monoclonal antibodies (mAbs) used, since until now there are not EC specific markers.…”

Section: Introductionmentioning

confidence: 99%

Mechanical Isolation of Human Endothelial Cells from Large Vessels for Flow Cytometry Immunophenotyping

Torres¹

2016

J Tissue Sci Eng

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Endothelial cells (EC) have important physiological functions, and they may also have a role in pathology. To better understand their role in health and disease, we must know very well their phenotype. Previous studies have identified and characterized EC mainly by immunohistochemistry, but there are also some studies using flow cytometry (FCM) after exposing these cells to enzymatic digestion, either to isolate and/ or to detach them from the vessel wall. However, it is well known that enzymatic treatment can cause deleterious effects on cell surface receptors, then influencing the antibody-antigen reaction. We describe a simple and cheap mechanical method to isolate EC from human vessels, avoiding alterations in the expression of cell surface receptors caused by the use of enzymes, and we tested it using FCM. With this method we were able to obtain fresh EC that were identified by FCM as a well-defined cluster of CD45 - CD146+bright CD31 +bright cells. This approach can be used in the future to isolate EC for further immunophenotypic characterization and for ex-vivo functional studies, as well as to test the effect of different stimuli, including pharmacological drugs.

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Flow cytometry: retrospective, fundamentals and recent instrumentation

Cited by 201 publications

References 60 publications

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

A novel allotetraploid gibel carp strain with maternal body type and growth superiority

Mechanical Isolation of Human Endothelial Cells from Large Vessels for Flow Cytometry Immunophenotyping

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