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A guide to choosing fluorescent protein combinations for flow cytometric analysis based on spectral overlap.

ABSTRACT: The advent of facile genome engineering technologies has made the generation of knock-in gene-expression or fusion-protein reporters more tractable. Fluorescent protein labeling of specific genes combined with surface marker profiling can more specifically identify a cell population. However, the question of which fluorescent proteins to utilize to generate reporter constructs is made difficult by the number of candidate proteins and the lack of updated experimental data on newer fluorescent proteins. Compounding this problem, most fluorescent proteins are designed and tested for use in microscopy. To address this, we cloned and characterized the detection sensitivity, spectral overlap, and spillover spreading of 13 monomeric fluorescent proteins to determine utility in multicolor panels. We identified a group of five fluorescent proteins with high signal to noise ratio, minimal spectral overlap, and low spillover spreading making them compatible for multicolor experiments. Specifically, generating reporters with combinations of three of these proteins would allow efficient measurements even at low-level expression. Because the proteins are monomeric, they could function either as gene-expression or as fusion-protein reporters. Additionally, this approach can be generalized as new fluorescent proteins are developed to determine their usefulness in multicolor panels. © 2018 International Society for Advancement of Cytometry.

SUBMITTER: Kleeman B

PROVIDER: S-EPMC8008483 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionClinicians and patients face a daunting task when choosing the most appropriate probiotic for their specific needs. Available preparations encompass a diverse and continuously expanding product base, with most available products lacking evidence-based trials that support their use. Even when evidence exists, not all probiotic products are equally effective for all disease prevention or treatment indications. At this point in time, drug regulatory agencies offer limited assistance with regard to guidance and oversight in most countries, including the U.S.MethodsWe reviewed the current medical literature and sources on the internet to survey the types of available probiotic products and to determine which probiotics had evidence-based efficacy data. Standard medical databases from inception to June 2018 were searched and discussions with experts in the field were conducted. We graded the strength of the evidence for probiotics having multiple, randomized controlled trials and developed a guide for the practical selection of current probiotic products for specific uses.ResultsWe found the efficacy of probiotic products is both strain-specific and disease-specific. Important factors involved in choosing the appropriate probiotic include matching the strain(s) with the targeted disease or condition, type of formulation, dose used and the source (manufacturing quality control and shelf-life). While we found many probiotic products lacked confirmatory trials, we found sufficient evidence for 22 different types of probiotics from 249 trials to be included. For example, several types of probiotics had strong evidence for the prevention of antibiotic-associated diarrhea [Saccharomyces boulardii I-745, a three-strain mixture (Lactobacillus acidophilus CL1285, L. casei Lbc80r, L. rhamnosus CLR2) and L. casei DN114001]. Strong evidence was also found for four types of probiotics for the prevention of a variety of other diseases/conditions (enteral-feed associated diarrhea, travellers' diarrhea, necrotizing enterocolits and side-effects associated with H. pylori treatments. The evidence was most robust for the treatment of pediatric acute diarrhea based on 59 trials (7 types of probiotics have strong efficacy), while an eight-strain multi-strain mixture showed strong efficacy for inflammatory bowel disease and two types of probiotics had strong efficacy for irritable bowel disease. Of the 22 types of probiotics reviewed, 15 (68%) had strong-moderate evidence for efficacy for at least one type of disease.ConclusionThe choice of an appropriate probiotic is multi-factored, based on the mode and type of disease indication and the specific efficacy of probiotic strain(s), as well as product quality and formulation.Trial registrationThis review was registered with PROSPERO: CRD42018103979.

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A guide to choosing fluorescent protein combinations for flow cytometric analysis based on spectral overlap.

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