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 Proteomic analysis of V-ATPase-rich cells harvested from the kidney and epididymis by fluorescence-activated cell sorting

2010 ◽  
Vol 298 (6) ◽  
pp. C1326-C1342 ◽  
Author(s):  
Nicolas Da Silva ◽  
Trairak Pisitkun ◽  
Clémence Belleannée ◽  
Lance R. Miller ◽  
Raoul Nelson ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Enzymatic Digestion ◽  
Potential Candidate ◽  
Fluorescence Activated Cell Sorting ◽  
Cytoskeletal Dynamics ◽  
Liquid Chromatography Tandem Mass ◽  
Green Fluorescent ◽  
Extracellular Environment

Proton-transporting cells are located in several tissues where they acidify the extracellular environment. These cells express the vacuolar H+-ATPase (V-ATPase) B1 subunit (ATP6V1B1) in their plasma membrane. We provide here a comprehensive catalog of the proteins that are expressed in these cells, after their isolation by enzymatic digestion and fluorescence-activated cell sorting (FACS) from transgenic B1-enhanced green fluorescent protein (EGFP) mice. In these mice, type A and B intercalated cells and connecting segment cells of the kidney, and narrow and clear cells of the epididymis, which all express ATP6V1B1, also express EGFP, while all other cell types are negative. The proteome of renal and epididymal EGFP-positive (EGFP+) cells was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and compared with their respective EGFP-negative (EGFP−) cell populations. A total of 2,297 and 1,564 proteins were detected in EGFP+ cells from the kidney and epididymis, respectively. Out of these proteins, 202 and 178 were enriched by a factor greater than 1.5 in EGFP+ cells compared with EGFP− cells, in the kidney and epididymis respectively, and included subunits of the V-ATPase (B1, a4, and A). In addition, several proteins involved in intracellular trafficking, signaling, and cytoskeletal dynamics were identified. A novel common protein that was enriched in renal and epididymal EGFP+ cells is the progesterone receptor, which might be a potential candidate for the regulation of V-ATPase-dependent proton transport. These proteomic databases provide a framework for comprehensive future analysis of the common and distinct functions of V-ATPase-B1-expressing cells in the kidney and epididymis.

Cell-sorting in aggregates of Dictyostelium discoideum

1999 ◽  
Vol 112 (22) ◽  
pp. 3923-3929 ◽  
Author(s):  
A. Nicol ◽  
W. Rappel ◽  
H. Levine ◽  
W.F. Loomis
Keyword(s):  
Cell Sorting ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Three Dimensional ◽  
Cell Movement ◽  
Time Lapse ◽  
Cell Types ◽  
Cell Type ◽  
Prespore Cell ◽  
Green Fluorescent

When Dictyostelium cells are induced to develop between a coverslip and a layer of agarose, they aggregate normally into groups containing up to a thousand cells but are then constrained to form disks only a few cells thick that appear to be equivalent to the three-dimensional mounds formed on top of agarose. Such vertically restricted aggregates frequently develop into elongated motile structures, the flattened equivalent of three-dimensional slugs. The advantage of using this system is that the restricted z-dimension enables direct microscopic visualization of most of the cells in the developing structure. We have used time lapse digital fluorescence microscopy of Dictyostelium strains expressing green fluorescent protein (GFP) under the control of either prestalk or prespore specific promoters to follow cell sorting in these flattened mounds. We find that prestalk and prespore cells expressing GFP arise randomly in early aggregates and then rotate rapidly around the disk mixed with the other cell type. After a few hours, the cell types sort out by a process which involves striking changes in relative cell movement. Once sorted, the cell types move independently of each other showing very little heterotypic adhesion. When a group of prestalk cells reaches the edge of the disk, it moves out and is followed by the prespore cell mass. We suggest that sorting may result from cell type specific changes in adhesion and the consequent disruption of movement in the files of cells that are held together by end-to-end adhesion.

scholarly journals Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform

2003 ◽  
Vol 370 (2) ◽  
pp. 429-438 ◽  
Author(s):  
Malcolm SHEPHERD ◽  
Theresa McSORLEY ◽  
Aileen E. OLSEN ◽  
Lee Ann JOHNSTON ◽  
Neil C. THOMSON ◽  
...  
Keyword(s):  
Tissue Distribution ◽  
Fluorescent Protein ◽  
Microscopic Analysis ◽  
Cell Types ◽  
Subcellular Fractionation ◽  
Pde4 Inhibitor ◽  
Specific Cell ◽  
Rnase Protection ◽  
Green Fluorescent ◽  
Camp Levels

We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of ‘long’ PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein—PDE4B4 chimaera. PDE4B4 exhibited a Km for cAMP of 5.4μM and a Vmax, relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone} with an IC50 of 83nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types.

scholarly journals Compartmentalized cyanophycin metabolism in the diazotrophic filaments of a heterocyst-forming cyanobacterium

2014 ◽  
Vol 111 (10) ◽  
pp. 3823-3828 ◽  
Author(s):  
Mireia Burnat ◽  
Antonia Herrero ◽  
Enrique Flores
Keyword(s):  
Fluorescent Protein ◽  
Cell Types ◽  
Oxygenic Photosynthesis ◽  
Specific Cell ◽  
Protein Fusion ◽  
Vegetative Cells ◽  
Polar Granules ◽  
Green Fluorescent ◽  
Multicellular Organisms ◽  
Cyanophycin Synthetase

Heterocyst-forming cyanobacteria are multicellular organisms in which growth requires the activity of two metabolically interdependent cell types, the vegetative cells that perform oxygenic photosynthesis and the dinitrogen-fixing heterocysts. Vegetative cells provide the heterocysts with reduced carbon, and heterocysts provide the vegetative cells with fixed nitrogen. Heterocysts conspicuously accumulate polar granules made of cyanophycin [multi-L-arginyl-poly (L-aspartic acid)], which is synthesized by cyanophycin synthetase and degraded by the concerted action of cyanophycinase (that releases β-aspartyl-arginine) and isoaspartyl dipeptidase (that produces aspartate and arginine). Cyanophycin synthetase and cyanophycinase are present at high levels in the heterocysts. Here we created a deletion mutant of geneall3922encoding isoaspartyl dipeptidase in the model heterocyst-forming cyanobacteriumAnabaenasp. strain PCC 7120. The mutant accumulated cyanophycin and β-aspartyl-arginine, and was impaired specifically in diazotrophic growth. Analysis of anAnabaenastrain bearing an All3922-GFP (green fluorescent protein) fusion and determination of the enzyme activity in specific cell types showed that isoaspartyl dipeptidase is present at significantly lower levels in heterocysts than in vegetative cells. Consistently, isolated heterocysts released substantial amounts of β-aspartyl-arginine. These observations imply that β-aspartyl-arginine produced from cyanophycin in the heterocysts is transferred intercellularly to be hydrolyzed, producing aspartate and arginine in the vegetative cells. Our results showing compartmentalized metabolism of cyanophycin identify the nitrogen-rich molecule β-aspartyl-arginine as a nitrogen vehicle in the unique multicellular system represented by the heterocyst-forming cyanobacteria.

scholarly journals Detection of Infectious Adenoviruses in Environmental Waters by Fluorescence-Activated Cell Sorting Assay

2010 ◽  
Vol 76 (5) ◽  
pp. 1442-1448 ◽  
Author(s):  
Dan Li ◽  
Miao He ◽  
Sunny C. Jiang
Keyword(s):  
Cell Sorting ◽  
Environmental Samples ◽  
Fluorescent Protein ◽  
Fluorescence Activated Cell Sorting ◽  
Treated Sewage ◽  
Serotype 5 ◽  
Seawater Samples ◽  
E1a Gene ◽  
Green Fluorescent ◽  
Public Health Protection

ABSTRACT Methods for rapid detection and quantification of infectious viruses in the environment are urgently needed for public health protection. A fluorescence-activated cell-sorting (FACS) assay was developed to detect infectious adenoviruses (Ads) based on the expression of viral protein during replication in cells. The assay was first developed using recombinant Ad serotype 5 (rAd5) with the E1A gene replaced by a green fluorescent protein (GFP) gene. Cells infected with rAd5 express GFP, which is captured and quantified by FACS. The results showed that rAd5 can be detected at concentrations of 1 to 104 PFU per assay within 3 days, demonstrating a linear correlation between the viral concentration and the number of GFP-positive cells with an r 2 value of >0.9. Following the same concept, FACS assays using fluorescently labeled antibodies specific to the E1A and hexon proteins, respectively, were developed. Assays targeting hexon showed greater sensitivity than assays targeting E1A. The results demonstrated that as little as 1 PFU Ads was detected by FACS within 3 days based on hexon protein, with an r 2 value greater than 0.9 over a 4-log concentration range. Application of this method to environmental samples indicated positive detection of infectious Ads in 50% of primary sewage samples and 33% of secondary treated sewage samples, but none were found in 12 seawater samples. The infectious Ads ranged in quantity between 10 and 165 PFU/100 ml of sewage samples. The results indicate that the FACS assay is a rapid quantification tool for detecting infectious Ads in environmental samples and also represents a considerable advancement for rapid environmental monitoring of infectious viruses.

scholarly journals Identification of Listeria monocytogenes In Vivo-Induced Genes by Fluorescence-Activated Cell Sorting

2001 ◽  
Vol 69 (8) ◽  
pp. 5016-5024 ◽  
Author(s):  
Rebecca L. Wilson ◽  
A. R. Tvinnereim ◽  
Bradley D. Jones ◽  
John T. Harty
Keyword(s):  
Listeria Monocytogenes ◽  
Cell Sorting ◽  
Fluorescent Protein ◽  
Fluorescence Activated Cell Sorting ◽  
Gfp Expression ◽  
Sorting Technique ◽  
J774 Cells ◽  
Severe Infections ◽  
Green Fluorescent

ABSTRACT Listeria monocytogenes is a gram-positive, intracellular, food-borne pathogen capable of causing severe infections in immunocompromised or pregnant individuals, as well as numerous animal species. Genetic analysis of Listeriapathogenesis has identified several genes which are crucial for virulence. The transcription of most of these genes has been shown to be induced upon entry of Listeria into the host cell. To identify additional genes that are induced in vivo and may be required for L. monocytogenes pathogenesis, a fluorescence-activated cell-sorting technique was initiated. Random fragments of the L. monocytogenes chromosome were cloned into a plasmid carrying a promoterless green fluorescent protein (GFP) gene, and the plasmids were transformed into the L. monocytogenes actA mutant DP-L1942. Fluorescence-activated cell sorting (FACS) was used to isolate L. monocytogenes clones that exhibited increased GFP expression within macrophage-like J774 cells but had relatively low levels of GFP expression when the bacteria were extracellular. Using this strategy, several genes were identified, including actA, that exhibited such an expression profile. In-frame deletions of two of these genes, one encoding the putative L. monocytogenesuracil DNA glycosylase (ung) and one encoding a protein with homology to the Bacillus subtilis YhdP hemolysin-like protein, were constructed and introduced into the chromosome of wild-type L. monocytogenes 10403s. TheL. monocytogenes 10403s ung deletion mutant was not attenuated for virulence in mice, while theyhdP mutant exhibited a three- to sevenfold reduction in virulence.

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