Highly Stable Red-Emissive Ratiometric Probe for Monitoring β-Galactosidase Activity Using Fluorescence Microscopy and Flow Cytometry

Routine methods for virus detection in clinical specimens rely on a variety of sensitive methods, such as genetic, cell culture and immuno-based assays. It is imperative that the detection assays would be reliable, reproducible, sensitive and rapid. Isolation of viruses from clinical samples is crucial for deeper virus identification and analysis. Here we introduce a rapid cell-based assay for isolation and detection of viruses. As a proof of concept several model viruses including West Nile Virus (WNV), Modified Vaccinia Ankara (MVA) and Adenovirus were chosen. Suspended Vero cells were employed to capture the viruses following specific antibody labeling which enables their detection by flow cytometry and immuno-fluorescence microscopy assays. Using flow cytometry, a dose response analysis was performed in which 3.6e4 pfu/mL and 1e6 pfu/mL of MVA and WNV could be detected within two hours, respectively. When spiked to commercial pooled human serum, detection sensitivity was slightly reduced to 3e6 pfu/mL for WNV, but remained essentially the same for MVA. In conclusion, the study demonstrates a robust and rapid methodology for virus detection using flow cytometry and fluorescence microscopy. We propose that this proof of concept may prove useful in identifying future pathogens.

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Thiazolidinedione toxicity to isolated hepatocytes revealed by coherent multiprobe fluorescence microscopy and correlated with multiparameter flow cytometry of peripheral leukocytes

Archives of Toxicology ◽

10.1007/s002040100251 ◽

2001 ◽

Vol 75 (7) ◽

pp. 425-438 ◽

Cited By ~ 51

Author(s):

Jeffrey Haskins ◽

Paul Rowse ◽

Ramin Rahbari ◽

Felix de la Iglesia

Keyword(s):

Flow Cytometry ◽

Fluorescence Microscopy ◽

Isolated Hepatocytes ◽

Multiparameter Flow Cytometry ◽

Peripheral Leukocytes

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.1.408-412.2003 ◽

2003 ◽

Vol 47 (1) ◽

pp. 408-412 ◽

Cited By ~ 21

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.

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Engineered viral nanoparticles for flow cytometry and fluorescence microscopy applications

Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology ◽

10.1002/wnan.1177 ◽

2012 ◽

Vol 4 (5) ◽

pp. 511-524 ◽

Cited By ~ 9

Author(s):

Kelly L. Robertson ◽

Jinny L. Liu

Keyword(s):

Flow Cytometry ◽

Fluorescence Microscopy ◽

Viral Nanoparticles

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Pannexin channels increase propidium iodide permeability in frozen–thawed dog spermatozoa

Reproduction Fertility and Development ◽

10.1071/rd16267 ◽

2017 ◽

Vol 29 (11) ◽

pp. 2269 ◽

Cited By ~ 1

Author(s):

J. L. Torres ◽

J. Palomino ◽

R. D. Moreno ◽

M. De los Reyes

Keyword(s):

Flow Cytometry ◽

Fluorescence Microscopy ◽

Analysis Of Variance ◽

Propidium Iodide ◽

Membrane Channels ◽

Flow Cytometry Data ◽

Viable Cells ◽

Head Segment ◽

Relationship Of ◽

The Relationship

Pannexins (Panx) are proteins that form functional single membrane channels, but they have not yet been described in dogs. The aim of the present study was to detect Panx1, Panx2 and Panx3 in frozen–thawed dog spermatozoa using flow cytometry and immunofluorescence analyses, evaluating the relationship of these proteins with propidium iodide (PI) in frozen–thawed spermatozoa. Fresh and frozen–thawed dog spermatozoa from eight dogs were preincubated with 3 μM PI with or without 15 μM carbenoxolone (CBX) or 1 mM probenecid (PBD), two Panx channel inhibitors, and then incubated with rabbit anti-Panx1, anti-Panx2 and anti-Panx3 antibodies (1 : 200). Panx immunolocalisation was assessed by fluorescence microscopy. Flow cytometry data were evaluated by analysis of variance. All three Panx proteins were found in dog spermatozoa: Panx1 was mostly localised to the acrosomal and equatorial segment, Panx2 was found in the posterior region of the head and tail and Panx3 was localised to the equatorial and posterior head segment. The percentage of PI-positive cells determined by flow cytometry was reduced (P < 0.05) in the presence of Panx inhibitors. These results show that Panx proteins are present in dog spermatozoa and increase PI permeability in frozen–thawed dog sperm, suggesting that the percentage of PI-positive spermatozoa used as an indicator of non-viable cells may lead to overestimation of non-viable cells.

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Actin polymerization and depolymerization during apoptosis in HL-60 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.6.c1981 ◽

1996 ◽

Vol 271 (6) ◽

pp. C1981-C1992 ◽

Cited By ~ 90

Author(s):

M. G. Levee ◽

M. I. Dabrowska ◽

J. L. Lelli ◽

D. B. Hinshaw

Keyword(s):

Flow Cytometry ◽

Fluorescence Microscopy ◽

Uv Irradiation ◽

Gel Electrophoresis ◽

Actin Polymerization ◽

Polyacrylamide Gel Electrophoresis ◽

Morphological Features ◽

Apoptotic Bodies ◽

Apoptotic Body ◽

Body Formation

Little is known about the biochemical “machinery” responsible for the morphological features of apoptosis, although the cytoskeleton is presumed to be involved. Using flow cytometry, polyacrylamide gel electrophoresis, and fluorescence microscopy, we show that apoptosis induced by ultraviolet (UV) irradiation or 80 micrograms/ml etoposide correlates with early transient polymerization and later depolymerization of filamentous (F)-actin and dramatic changes in visible microfilament organization. Depolymerization of F-actin began before the formation of apoptotic bodies and was ultimately composed of decreases in both the detergent-insoluble (40%) and detergent-soluble (50%) pools of F-actin. Dihydrocytochalasin B (H2CB), which blocked apoptotic body formation, depolymerized F-actin in the detergent-insoluble pool only. Visually, H2CB treatment disrupted microfilament organization, resulting in short, brightly stained microfilaments dispersed throughout the cytoplasm. In contrast, apoptotic cells contained a network of fine microfilaments with bright staining concentrated at the site of apoptotic body formation. Together, these results suggest that reorganization of the microfilament network is necessary for the formation of apoptotic bodies and that depolymerization of F-actin may also be a necessary component of the process of apoptosis.

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Detection of terminal deoxynucleotidyl transferase (TdT) by flow cytometry in leukemic disorders.

Journal of Histochemistry & Cytochemistry ◽

10.1177/37.4.2647840 ◽

1989 ◽

Vol 37 (4) ◽

pp. 509-513 ◽

Cited By ~ 11

Author(s):

R H Bardales ◽

A Carrato ◽

M Fleischer ◽

M K Schwartz ◽

B Koziner

Keyword(s):

Flow Cytometry ◽

Fluorescence Microscopy ◽

Myeloid Leukemia ◽

De Novo ◽

Lymphoblastic Leukemia ◽

Biochemical Assay ◽

Terminal Deoxynucleotidyl Transferase ◽

Large Numbers ◽

A New Technique ◽

T Lymphoblastic Lymphoma

We applied a new technique to the detection of intracellular TdT in 26 leukemic patients, including 16 non-T acute lymphoblastic leukemia (ALL), four T-ALL, one T-lymphoblastic lymphoma in leukemia phase, one undifferentiated leukemia, one de novo lymphoblastic phase of chronic myeloid leukemia, and three acute monocytic leukemias (AMOL). Mononuclear cell suspensions were incubated in saponin to permeabilize the cell membrane. The cells were then stained by indirect immunofluorescence (IF) using anti-human TdT monoclonal antibodies and were analyzed by flow cytometry (FCM). The TdT results were compared with those obtained by biochemical TdT assay (26 cases), immunoperoxidase determination (PAP) (12 cases), and fluorescence microscopy (seven cases). The results obtained by PAP and fluorescence microscopy were 100% concordant with those obtained by FCM and biochemical assay. TdT determination by FCM allows the analysis of large numbers of cells in a fast, objective, and reliable manner, as compared with biochemical assay, PAP, and fluorescence microscopy determinations.

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