Fluorescence Activated Cell Sorting of Shoot Apical Meristem Cell Types

2013 ◽  
pp. 315-321 ◽  
Author(s):  
G. Venugopala Reddy
Keyword(s):  
Cell Sorting ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Cell Types ◽  
Fluorescence Activated Cell Sorting ◽  
Meristem Cell

Purification of functional lactotrophs and somatotrophs from female rats using fluorescence-activated cell sorting

1990 ◽  
Vol 126 (2) ◽  
pp. 269-274 ◽  
Author(s):  
D. Wynick ◽  
R. Critchley ◽  
M. S. Venetikou ◽  
J. M. Burrin ◽  
S. R. Bloom
Keyword(s):  
Cell Surface ◽  
Cell Sorting ◽  
Anterior Pituitary ◽  
Cell Types ◽  
Female Rats ◽  
Normal Female ◽  
Pituitary Cells ◽  
Female Rat ◽  
Fluorescence Activated Cell Sorting ◽  
Surface Labelling

ABSTRACT As the secretory granules of anterior pituitary cells fuse with the cell surface, there would appear to be sufficient hormone present on the cell surface to be labelled by polyclonal hormone antibodies and thus analysed by flow cytometry. We have therefore applied fluorescence-activated cell sorting to these labelled pituitary cells. Percentage purity and depletion of other cell types was assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96·7 ±1·7 (s.e.m.)% and 98±1·0% respectively by immunocytochemistry, and to 95·8 ±1·1% and 97±0·8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 ±3·4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Separating cells on the basis of the intensity of prolactin cell-surface labelling demonstrated a low level of binding of the prolactin antibody to gonadotrophs (but not of sufficient fluorescence intensity to be sorted into the prolactin enriched population), raising the possibility of prolactin receptors on gonadotrophs. We were unable to demonstrate the presence of mammosomatotrophs in the normal female rat, since purified lactotrophs did not contain or secrete GH nor did purified somatotrophs contain or secrete prolactin. Journal of Endocrinology (1990) 126, 269–274

scholarly journals Roles of plant growth regulators on flowering of rose (Rosa hybrida L.’Red Rose’)

2021 ◽  
Vol 947 (1) ◽  
pp. 012039
Author(s):  
Linh Tran Minh Hong ◽  
Tu Cam Trinh ◽  
Viet Trang Bui ◽  
Huong Thanh Tran
Keyword(s):  
Plant Growth ◽  
Plant Growth Regulators ◽  
Growth Regulators ◽  
Shoot Apical Meristem ◽  
Apical Meristem ◽  
Rosa Hybrida ◽  
Floral Meristem ◽  
Flower Bud ◽  
Meristem Cell

Abstract Rose is the most popular ornamental flower all over the world, which is used as garden plants and cut flowers. In the case of Rosa hybrida L. ’Red Rose’, flowering provides the major developmental transition from the vegetative to the reproductive stage, and reproduction is one of the most important phases in an organism’s life cycle. In this study, the morphological and physiological changes during the flower development of rose, which is planted in the garden, and roles of plant growth regulators on the flowering of in vitro vegetative shoots of rose were analyzed. The development of a flower includes three stages: the shoot apical meristem, floral meristem, floral bud. Levels of cytokinin, auxins, and gibberellins increased in the transition of meristem from the shoot apical meristem to the floral meristem stage. Plant growth regulators have important effects on the shoot apical meristem cell division and flowering. The combination of 0.5 mg.L−1 GA3, 0.1 mg.L−1 NAA, 2.5 or 3.0 mg.L−1 BA to Murashige and Skoog (MS) medium induces the floral transition of the in vitro vegetative shoots with the highest percentage (41%) as well as growth and development in comparison to the other treatments after 10 weeks. Then, the in vitro floral meristem continuously developed into a flower bud after 12 weeks.

scholarly journals Fluorescence-activated cell sorting to reveal the cell origin of radioligand binding

2019 ◽  
Vol 40 (6) ◽  
pp. 1242-1255 ◽  
Author(s):  
Benjamin B Tournier ◽  
Stergios Tsartsalis ◽  
Kelly Ceyzériat ◽  
Zadith Medina ◽  
Ben H Fraser ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Sorting ◽  
Radioligand Binding ◽  
Single Photon ◽  
Photon Emission ◽  
Cell Types ◽  
Translocator Protein ◽  
Emission Computed Tomography ◽  
Fluorescence Activated Cell Sorting

Many studies have explored the role of TSPO (18 kDa translocator protein) as a marker of neuroinflammation using single-photon emission computed tomography (SPECT) or positron emission tomography (PET). In vivo imaging does not allow to determine the cells in which TSPO is altered. We propose a methodology based on fluorescence-activated cell sorting to sort different cell types of radioligand-treated tissues. We compared left/right hippocampus of rats in response to a unilateral injection of lipopolysaccharide (LPS), ciliary neurotrophic factor (CNTF) or saline. We finally applied this methodology in human samples (Alzheimer's disease patients and controls). Our data show that the pattern of TSPO overexpression differs across animal models of acute neuroinflammation. LPS induces a microglial expansion and an increase in microglial TSPO binding. CNTF is associated with an increase in TSPO binding in microglia and astrocytes in association with an increase in the number of microglial binding sites per cell. In humans, we show that the increase in CLINDE binding in Alzheimer's disease concerns microglia and astrocytes in the presence of a microglial expansion. Thus, the cellular basis of TSPO overexpression is condition dependent, and alterations in TSPO binding found in PET/SPECT imaging studies cannot be attributed to particular cell types indiscriminately.

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.

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.

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