A Two‐Stage Flow Cytometry Strategy to Distinguish Single Cells from Doublets in Heterogeneous Cell Mixtures and Improve Cell Cluster Identification: Application to Human Monocyte Subpopulations

2021 ◽  
Vol 1 (8) ◽  
Author(s):  
Fadila Merah‐Mourah ◽  
Shannon O. Cohen ◽  
Alain Haziot
Keyword(s):  
Flow Cytometry ◽  
Single Cells ◽  
Human Monocyte ◽  
Cell Cluster ◽  
Two Stage ◽  
Cluster Identification ◽  
Monocyte Subpopulations

scholarly journals Cellular lensing and near infrared fluorescent nanosensor arrays to enable chemical efflux cytometry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Soo-Yeon Cho ◽  
Xun Gong ◽  
Volodymyr B. Koman ◽  
Matthias Kuehne ◽  
Sun Jin Moon ◽  
...  
Keyword(s):  
Near Infrared ◽  
Single Cells ◽  
Microfluidic Channel ◽  
Human Monocyte ◽  
Cellular Heterogeneity ◽  
Label Free ◽  
Nanotube Array ◽  
Chemical Cytometry ◽  
Rich Information ◽  
Non Destructive

AbstractNanosensors have proven to be powerful tools to monitor single cells, achieving spatiotemporal precision even at molecular level. However, there has not been way of extending this approach to statistically relevant numbers of living cells. Herein, we design and fabricate nanosensor array in microfluidics that addresses this limitation, creating a Nanosensor Chemical Cytometry (NCC). nIR fluorescent carbon nanotube array is integrated along microfluidic channel through which flowing cells is guided. We can utilize the flowing cell itself as highly informative Gaussian lenses projecting nIR profiles and extract rich information. This unique biophotonic waveguide allows for quantified cross-correlation of biomolecular information with various physical properties and creates label-free chemical cytometer for cellular heterogeneity measurement. As an example, the NCC can profile the immune heterogeneities of human monocyte populations at attomolar sensitivity in completely non-destructive and real-time manner with rate of ~600 cells/hr, highest range demonstrated to date for state-of-the-art chemical cytometry.

Fast intracellular pH determination in single cells by flow-cytometry

1981 ◽  
Vol 68 (5) ◽  
pp. 265-266 ◽  
Author(s):  
G. Valet ◽  
A. Raffael ◽  
L. Moroder ◽  
E. W�nsch ◽  
G. Ruhenstroth-Bauer
Keyword(s):  
Flow Cytometry ◽  
Intracellular Ph ◽  
Single Cells

scholarly journals Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0240769
Author(s):  
Prasanna Channathodiyil ◽  
Jonathan Houseley
Keyword(s):  
Flow Cytometry ◽  
Transcriptome Analysis ◽  
Mammalian Cells ◽  
Single Cells ◽  
Rna Extraction ◽  
Cyclin B1 ◽  
Immunofluorescent Staining ◽  
Rna Seq ◽  
Simple Method ◽  
Staining Methods

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

scholarly journals Differential interleukin-1 elaboration by density-defined human monocyte subpopulations

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 298-301
Author(s):  
JA Elias ◽  
P Chien ◽  
KM Gustilo ◽  
AD Schreiber
Keyword(s):  
Human Blood ◽  
Interleukin 1 ◽  
Human Monocyte ◽  
Mononuclear Phagocytes ◽  
Blood Monocyte ◽  
Thymocyte Proliferation ◽  
Percoll Gradients ◽  
Human Blood Monocyte ◽  
Monocytes And Macrophages ◽  
Monocyte Subpopulations

Interleukin-1 (IL-1) is an important immunoregulatory peptide produced by monocytes and macrophages. Because mononuclear phagocytes are morphologically and functionally heterogeneous, we examined whether they differ in their ability to elaborate IL-1. We used discontinuous Percoll gradients to obtain five density-defined human blood monocyte subpopulations. Unfractionated monocytes and their subsets were compared for their ability to stimulate thymocyte proliferation. Supernatants obtained from the denser monocytes consistently contained more IL-1 activity than did supernatants from the less dense cells. This difference in IL-1 activity was the result of differences in IL-1 elaboration, not the selective production of an inhibitor of IL-1- induced thymocyte proliferation. These data demonstrate that density- defined human monocyte subpopulations differ in their capacity to elaborate IL-1.

scholarly journals Differential interleukin-1 elaboration by density-defined human monocyte subpopulations

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 298-301 ◽  
Author(s):  
JA Elias ◽  
P Chien ◽  
KM Gustilo ◽  
AD Schreiber
Keyword(s):  
Human Blood ◽  
Interleukin 1 ◽  
Human Monocyte ◽  
Mononuclear Phagocytes ◽  
Blood Monocyte ◽  
Thymocyte Proliferation ◽  
Percoll Gradients ◽  
Human Blood Monocyte ◽  
Monocytes And Macrophages ◽  
Monocyte Subpopulations

Abstract Interleukin-1 (IL-1) is an important immunoregulatory peptide produced by monocytes and macrophages. Because mononuclear phagocytes are morphologically and functionally heterogeneous, we examined whether they differ in their ability to elaborate IL-1. We used discontinuous Percoll gradients to obtain five density-defined human blood monocyte subpopulations. Unfractionated monocytes and their subsets were compared for their ability to stimulate thymocyte proliferation. Supernatants obtained from the denser monocytes consistently contained more IL-1 activity than did supernatants from the less dense cells. This difference in IL-1 activity was the result of differences in IL-1 elaboration, not the selective production of an inhibitor of IL-1- induced thymocyte proliferation. These data demonstrate that density- defined human monocyte subpopulations differ in their capacity to elaborate IL-1.

scholarly journals Pattern of human monocyte subpopulations in health and disease

2020 ◽  
Vol 92 (1) ◽  
Author(s):  
Agnieszka Ożańska ◽  
Donata Szymczak ◽  
Justyna Rybka
Keyword(s):  
Human Monocyte ◽  
Health And Disease ◽  
Monocyte Subpopulations

scholarly journals Flow Cytometric Assessment of Susceptibilities of Streptococcus pyogenes to Erythromycin and Rokitamycin

2003 ◽  
Vol 47 (1) ◽  
pp. 408-412 ◽  
Author(s):  
Pier Carlo Braga ◽  
Cinzia Bovio ◽  
Maria Culici ◽  
Monica Dal Sasso
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Streptococcus Pyogenes ◽  
Single Cells ◽  
Membered Ring ◽  
Flow Cytometric ◽  
Live Bacteria ◽  
Erythromycin A ◽  
Set Up ◽  
Syto 9

ABSTRACT The effects of erythromycin (a 14-membered ring macrolide) and rokitamycin (a 16-membered ring macrolide) on the viability of the Streptococcus pyogenes M phenotype were studied by means of flow cytometry and fluorescence microscopy by using a combination of two fluorochromes (syto 9 and propidium iodide) that stains live bacteria green and dead bacteria red. In order to apply the flow cytometry, a bacterial sonication procedure was expressly set up to separate single cells from the long, intralaced S. pyogenes chains of up to 30 to 40 cells that have previously prevented the application of flow cytometry to this type of bacteria. The association of flow cytometry using an appropriate sonication procedure, together with a combination of fluorescent probes, offered the possibility of very quickly investigating the different microbiological effects of rokitamycin at 2 μg/ml, which was active on the S. pyogenes M phenotype, and of erythromycin at doses of up to 32 μg/ml, which was not.

Differences in superoxide production by nonmigrating and migrating human monocyte subpopulations

1982 ◽  
Vol 108 (1) ◽  
pp. 392-398 ◽  
Author(s):  
Liana Harvath ◽  
Janis K. Lazdins ◽  
Enrica Alteri ◽  
Edward J. Leonard
Keyword(s):  
Human Monocyte ◽  
Superoxide Production ◽  
Monocyte Subpopulations

A Two-Stage Dissociation System for Multilayer Imaging of Cancer Biomarker-Synergic Networks in Single Cells

2017 ◽  
Vol 129 (17) ◽  
pp. 4880-4883 ◽  
Author(s):  
Ruo-Can Qian ◽  
Yue Cao ◽  
Li-Jun Zhao ◽  
Zhen Gu ◽  
Yi-Tao Long
Keyword(s):  
Single Cells ◽  
Cancer Biomarker ◽  
Two Stage
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