scholarly journals A quantitative single-cell assay for retrograde membrane traffic enables rapid detection of defects in cellular organization

2020 ◽  
Vol 31 (7) ◽  
pp. 511-519 ◽  
Author(s):  
Phi Luong ◽  
Qian Li ◽  
Pin-Fang Chen ◽  
Paul J. Wrighton ◽  
Denis Chang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endoplasmic Reticulum ◽  
Single Cell ◽  
Membrane Trafficking ◽  
Rapid Detection ◽  
Cell Function ◽  
Cellular Organization ◽  
Intracellular Protein ◽  
Flow Cytometry Assay ◽  
Cell Flow Cytometry

Retrograde membrane trafficking from plasma membrane to the Golgi and endoplasmic reticulum affects intracellular protein dynamics underlying cell function. Here, we developed split-fluorescent toxin reporters that enable a quantitative, sensitive, and real-time single-cell flow cytometry assay for retrograde membrane transport.

scholarly journals Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria

BIO-PROTOCOL ◽  
2016 ◽  
Vol 6 (23) ◽  
Author(s):  
Monica Benincasa ◽  
Quentin Barrière ◽  
Giulia Runti ◽  
Olivier Pierre ◽  
Mick Bourge ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
Flow Cytometry Assay ◽  
Cell Flow Cytometry ◽  
Peptide Uptake

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

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.

Application of single-cell real-time imaging flow cytometry in rapid detection of pathogenic fungi in clinical liquid specimens

2021 ◽  
Vol 9 (2) ◽  
pp. 025004
Author(s):  
Linting Lv ◽  
Li Dong ◽  
Jiajia Zheng ◽  
Tuohutaerbieke Maermaer ◽  
Xiangbo Huang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
Real Time ◽  
Rapid Detection ◽  
Pathogenic Fungi ◽  
Real Time Imaging ◽  
Imaging Flow Cytometry

Single cell flow cytometry and machine learning stratifies patients undergoing transurethral resection of the bladder tumor (TURBT)

2019 ◽  
Vol 18 (2) ◽  
pp. e2406-e2407
Author(s):  
A. Koladiya ◽  
K. Otavová ◽  
V. Adamcová ◽  
J. Stejskal ◽  
B. Ogan ◽  
...  
Keyword(s):  
Machine Learning ◽  
Flow Cytometry ◽  
Single Cell ◽  
Transurethral Resection ◽  
Bladder Tumor ◽  
Cell Flow Cytometry

Adaptive genetic algorithms combined with high sensitivity single cell-based technology derived urine-based score to differentiate between high-grade and low-grade transitional cell carcinoma of the bladder.

2020 ◽  
Vol 38 (6_suppl) ◽  
pp. 572-572
Author(s):  
Shaheen Riadh Alanee ◽  
Zade Roumayah ◽  
Musatafa Deebajah ◽  
James O. Peabody ◽  
Rodrigo Mora ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Bladder Cancer ◽  
Single Cell ◽  
Transitional Cell ◽  
Low Grade ◽  
High Grade ◽  
Median Score ◽  
Adaptive Genetic Algorithms ◽  
Cell Flow Cytometry ◽  
Carcinoma Of The Bladder

572 Background: We previously showed that adaptive genetic algorithms (AGA), in combination with single-cell flow cytometry technology, can be used to develop a noninvasive urine-based score to detect bladder cancer with high accuracy. Our aim in this analysis was to investigate if that same score can differentiate between high grade (HG) and low grade (LG) transitional cell carcinoma of the bladder (BC). Methods: We collected urine samples from cystoscopy confirmed HG and LG superficial bladder cancer patients and healthy donors in an optimized urine collection media. We then examined these samples using an assay developed from AGA in combination with single-cell flow cytometry technology. Results: We examined 50 BC and 15 healthy donor urine samples. Patients were majorly White (59.2%), males (61.2%), and had HG BC (66.7%). AGA derived score of 1.1 differentiated between BCa and healthy patients with high precision (AUC 0.92). The median score was 2.8 for LG BC and 6 for LG BC. Mann-Whitney Rank Sum Test indicated that the difference between the median score of HG and LG BC was significant at P value = 0.003. The score performed well independent of patients’ sex or smoking history. Conclusions: Using single-cell technology and machine learning, we developed a new urine-based score that can potentially differentiate between HG and LG bladder cancer. Future studies are planned to validate this score.

Development, application and computational analysis of high-dimensional fluorescent antibody panels for single-cell flow cytometry

2019 ◽  
Vol 14 (7) ◽  
pp. 1946-1969 ◽  
Author(s):  
Jolanda Brummelman ◽  
Claudia Haftmann ◽  
Nicolás Gonzalo Núñez ◽  
Giorgia Alvisi ◽  
Emilia M. C. Mazza ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
Computational Analysis ◽  
Fluorescent Antibody ◽  
High Dimensional ◽  
Cell Flow Cytometry ◽  
Development Application

Single-Cell Flow Cytometry Profiling of BAL in Children

2020 ◽  
Vol 63 (2) ◽  
pp. 152-159
Author(s):  
Shivanthan Shanthikumar ◽  
Matthew Burton ◽  
Richard Saffery ◽  
Sarath C. Ranganathan ◽  
Melanie R. Neeland
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
Cell Flow Cytometry

scholarly journals IBEX – A versatile multi-plex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues

2020 ◽  
Author(s):  
Andrea J. Radtke ◽  
Evelyn Kandov ◽  
Bradley Lowekamp ◽  
Emily Speranza ◽  
Colin J. Chu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
Cell Types ◽  
Spatial Context ◽  
Tissue Imaging ◽  
Imaging Data ◽  
Fine Grained ◽  
Sequencing Technologies ◽  
Molecular Features ◽  
Cell Flow Cytometry

AbstractThe diverse composition of mammalian tissues poses challenges for understanding the cell-cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only 2-6 molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a new method that overcomes major impediments to highly multi-plex tissue imaging. Iterative Bleaching Extends multi-pleXity (IBEX) uses an iterative staining and chemical bleaching method to enable high resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an ‘open’ system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and IBEX analysis of the same tissue. To facilitate data processing, we provide an open source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high content imaging to reveal the complex cellular landscape of diverse organs and tissues.Significance StatementSingle cell flow cytometry and genomic methods are rapidly increasing our knowledge of the diversity of cell types in metazoan tissues. However, suitably robust methods for placing these cells in a spatial context that reveal how their localization and putative interactions contribute to tissue physiology and pathology are still lacking. Here we provide a readily accessible pipeline (IBEX) for highly multi-plex immunofluorescent imaging that enables a fine-grained analysis of cells in their tissue context. Additionally, we describe extensions of the IBEX workflow to handle hard to image tissue preparations and a method to facilitate direct integration of the imaging data with flow cytometry and sequencing technologies.

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