A toehold-mediated strand displacement cascade-based DNA assay method via flow cytometry and magnetic separation

The aim of this study was to identify uterine pluripotent cells both in bovine uterine tissues as well in epithelial, stromal, and myometrial uterine cell populations. Moreover, the relationship of pluripotent markers expression with age and the uterine horn side was considered. Uterine tissue was collected from ipsilateral and contralateral horns (days 8–10 of the estrous cycle). Immunohistostaining for C-KIT, OCT3/4, NANOG, and SOX2 in uterine tissue was determined. mRNA expression of C-KIT, OCT3/4, NANOG and SOX2 was evaluated in uterine tissue relative to the age of the cow and uterine horn side. Gene and protein expression of these markers in the uterine luminal epithelial, stromal, and myometrial cells was evaluated by real-time PCR and western blotting respectively. The expression of pluripotent cell markers OCT3/4, NANOG, and SOX2 was identified by flow cytometry assay in epithelial, stromal, and myometrial cells. Multilineage differentiation of the bovine uterine cells was performed. mRNA expression of OCT3/4, NANOG, and SOX2 in uterine tissue was higher in the ipsilateral horn than in the contralateral horn. Flow cytometry assay revealed positive fluorescence for OCT3/4, NANOG, and SOX2 in all uterine cell types. Results showed the age-dependent expression of pluripotent markers in uterine tissue. Beside, the different expression of pluripotent cells in each horn of uterus suggests the influence of ovarian hormones on these characteristics. The highest mRNA and protein expression for pluripotent markers was observed in stromal cells among uterine cells, which indicates this population of cells as the main site of pluripotent cells in the cow uterus.

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Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

PLoS ONE ◽

10.1371/journal.pone.0133769 ◽

2015 ◽

Vol 10 (8) ◽

pp. e0133769 ◽

Cited By ~ 8

Author(s):

Wiltrud Haaß ◽

Helga Kleiner ◽

Martin C. Müller ◽

Wolf-Karsten Hofmann ◽

Alice Fabarius ◽

...

Keyword(s):

Flow Cytometry ◽

Proteolytic Activity ◽

Living Cells ◽

Flow Cytometry Assay ◽

Single Living Cells ◽

Single Living

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Single-Tube Flow Cytometry Assay for the Detection of Mature Lymphoid Neoplasms in Paucicellular Samples

Acta Cytologica ◽

10.1159/000448799 ◽

2016 ◽

Vol 60 (4) ◽

pp. 385-394

Author(s):

Alessandra Stacchini ◽

Anna Demurtas ◽

Sabrina Aliberti ◽

Antonella Barreca ◽

Domenico Novero ◽

...

Keyword(s):

Flow Cytometry ◽

Hodgkin Lymphoma ◽

Final Diagnosis ◽

Molecular Data ◽

Malignant Lymphomas ◽

Flow Cytometry Assay ◽

Single Tube ◽

Fine Needle Aspirates ◽

Non Hodgkin Lymphoma ◽

A Cell

Objectives: Flow cytometry (FC) has become a useful support for cytomorphologic evaluation (CM) of fine-needle aspirates (FNA) and serous cavity effusions (SCE) in cases of suspected non-Hodgkin lymphoma (NHL). FC results may be hampered by the scarce viability and low cellularity of the specimens. Study Design: We developed a single-tube FC assay (STA) that included 10 antibodies cocktailed in 8-color labeling, a cell viability dye, and a logical gating strategy to detect NHL in hypocellular samples. The results were correlated with CM and confirmed by histologic or molecular data when available. Results: Using the STA, we detected B-type NHL in 31 out of 103 hypocellular samples (81 FNA and 22 SCE). Of these, 8 were not confirmed by CM and 2 were considered to be only suspicious. The FC-negative samples had a final diagnosis of benign/reactive process (42/72), carcinoma (27/72), or Hodgkin lymphoma (3/72). Conclusions: The STA approach allowed obtainment of maximum immunophenotyping data in specimens containing a low number of cells and a large amount of debris. The information obtained by STA can help cytomorphologists not only to recognize but also to exclude malignant lymphomas.

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A flow cytometry assay to quantify intercellular exchange of membrane components

Chemical Science ◽

10.1039/c7sc00260b ◽

2017 ◽

Vol 8 (8) ◽

pp. 5585-5590 ◽

Cited By ~ 3

Author(s):

Dimitrios Poulcharidis ◽

Kimberley Belfor ◽

Alexander Kros ◽

Sander I. van Kasteren

Keyword(s):

Flow Cytometry ◽

Flow Cytometry Assay ◽

Membrane Components ◽

Intercellular Exchange

A technically simple and broadly deployable FACS-based assay to determine intercellular exchange of membrane components.

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Endoplasmic Reticulum Stress Is Involved in Glucocorticoid-Induced Apoptosis in PC12 Cells

Analytical Cellular Pathology ◽

10.1155/2021/5565671 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Shanyong Yi ◽

Weibo Shi ◽

Min Zuo ◽

Songjun Wang ◽

Rufei Ma ◽

...

Keyword(s):

Flow Cytometry ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Pc12 Cells ◽

Neuronal Injury ◽

Immunofluorescence Staining ◽

High Concentration ◽

Flow Cytometry Assay ◽

Apoptosis Rate ◽

Amino Terminal

Objective. The present study selected PC12 cells to construct a neuronal injury model induced by glucocorticoids (GC) in vitro, aiming to explore whether the endoplasmic reticulum stress (ERS) PKR-like endoplasmic reticulum kinase (PERK)-activating transcription factor 4 (ATF4)-C/EBP-homologous protein (CHOP) and inositol requirement 1 (IRE1)-apoptosis signal regulating kinase 1 (ASK1)-C-Jun amino-terminal kinase (JNK) signaling pathways are associated with the neuronal injury process induced by GC and provide morphological evidence. Methods. Cell models with different doses and different durations of GC exposure were established. The viability of PC12 cells was detected by the CCK-8 assay, and the apoptosis rate of PC12 cells was detected by the flow cytometry assay. The expression of microtubule-associated protein 2 (Map2); glucocorticoids receptor (GR); cellular oncogene fos (C-fos); and ERS-related proteins, glucose-regulated protein 78 (GRP78), p-PERK, p-IRE1, ATF4, ASK1, JNK, and CHOP, was observed by immunofluorescence staining. Results. The results of immunofluorescence staining showed that PC12 cells abundantly expressed Map2 and GR. The CCK-8 assay revealed that high-concentration GC exposure significantly inhibited the cell viability of PC12 cells. The flow cytometry assay indicated that high-concentration GC exposure significantly increased the apoptosis rate of PC12 cells. Immunofluorescence staining showed that GC exposure significantly increased the expression of C-fos, GRP78, p-PERK, p-IRE1, ATF4, ASK1, JNK, and CHOP. Treatment with ERS inhibitor 4-phenylbutyric acid (4-PBA) and GR inhibitor RU38486 attenuated related damage and downregulated the expression of the abovementioned proteins. Conclusion. High-concentration GC exposure can significantly inhibit the viability of PC12 cells and induce apoptosis. PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways are involved in the above damage process.

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Enzyme-free homogeneous electrochemical biosensor for DNA assay using toehold-triggered strand displacement reaction coupled with host-guest recognition of Fe3O4@SiO2@β-CD nanocomposites

Biosensors and Bioelectronics ◽

10.1016/j.bios.2018.04.035 ◽

2018 ◽

Vol 114 ◽

pp. 37-43 ◽

Cited By ~ 22

Author(s):

Jingjing Jiang ◽

Xinyi Lin ◽

Dong Ding ◽

Guowang Diao

Keyword(s):

Electrochemical Biosensor ◽

Displacement Reaction ◽

Strand Displacement ◽

Dna Assay ◽

Strand Displacement Reaction

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Flow cytometry with a monoclonal antibody to the low density lipoprotein receptor compared with gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia

Clinical Chemistry ◽

10.1093/clinchem/44.5.966 ◽

1998 ◽

Vol 44 (5) ◽

pp. 966-972 ◽

Cited By ~ 6

Author(s):

Bent Raungaard ◽

Finn Heath ◽

Jens Uffe Brorholt-Petersen ◽

Henrik Kjærulf Jensen ◽

Ole Faergeman

Keyword(s):

Flow Cytometry ◽

T Lymphocytes ◽

Receptor Gene ◽

Low Density Lipoprotein ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Receptor Expression ◽

Low Density ◽

Flow Cytometry Assay ◽

Stop Mutation

Abstract We used a fluorescence flow cytometry assay with a monoclonal low density lipoprotein (LDL) receptor-specific antibody to detect LDL receptor expression on blood T lymphocytes and monocytes. We prepared peripheral blood mononuclear cells from patients with genetically verified LDL receptor-defective (Trp66-Gly mutation, n = 17) or receptor-negative (Trp23-stop mutation, n = 17) heterozygous familial hypercholesterolemia (FH) and from healthy individuals (n = 24). The cells were stimulated to express the maximum amount of LDL receptor by preincubation in lipoprotein-deficient medium. A dual-labeling technique allowed flow cytometric analysis of LDL receptor expression on cells identified by fluorescently conjugated surface marker antibodies. Knowing the LDL receptor gene mutation of the FH patients allowed us to compare the diagnostic capability of this functional assay with the DNA diagnosis and to validate the assay with molecular genetics instead of clinical indices of heterozygous FH. T lymphocytes expressed more LDL receptors and gave better diagnostic results than monocytes, and cells from patients with either the Trp66-Gly or the Trp23-stop mutation had variable but significantly reduced LDL receptor expression. The data indicate that this fluorescence flow cytometry assay is unsuitable for diagnosis of individual cases of heterozygous FH but that it may be useful for functionally characterizing mutations in the LDL receptor gene.

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