scholarly journals Quantitative Flow Cytometric Evaluation of MaximalCryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model

2000 ◽  
Vol 66 (10) ◽  
pp. 4315-4317 ◽  
Author(s):  
Agnès Delaunay ◽  
Gilles Gargala ◽  
Xunde Li ◽  
Loic Favennec ◽  
Jean Jacques Ballet
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Risk Evaluation ◽  
Detection Sensitivity ◽  
Contaminated Water ◽  
Suckling Mouse ◽  
Flow Cytometric ◽  
Waterborne Transmission ◽  
Flow Cytometric Evaluation

ABSTRACT The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.

Flow cytometric evaluation of bladder cancer: recommendations of the NCI flow cytometry network for bladder cancer

1992 ◽  
Vol 10 (1) ◽  
pp. 63-67 ◽  
Author(s):  
R. L. Aamodt ◽  
J. S. Coon ◽  
A. Deitch ◽  
R. W. deVere White ◽  
L. G. Koss ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Bladder Cancer ◽  
Flow Cytometric ◽  
Flow Cytometric Evaluation

scholarly journals CD161 Is Expressed in a Subset of T-Cell Prolymphocytic Leukemia Cases and Is Useful for Disease Follow-up

2019 ◽  
Vol 152 (4) ◽  
pp. 471-478
Author(s):  
Scott R Gilles ◽  
Sophia L Yohe ◽  
Michael A Linden ◽  
Michelle Dolan ◽  
Betsy Hirsch ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Nk Cells ◽  
Retrospective Review ◽  
T Cell Subsets ◽  
Prolymphocytic Leukemia ◽  
Flow Cytometry Data ◽  
Flow Cytometric ◽  
Flow Cytometric Evaluation

AbstractObjectivesCD161 (NKRP1) is a lectin-like receptor present on NK cells and rare T-cell subsets. We have observed CD161 expression in some cases of T-cell prolymphocytic leukemia (T-PLL) and found it to be useful in follow-up and detection of disease after treatment.MethodsRetrospective review of T-PLL cases with complete flow cytometry data including CD161.ResultsWe identified 10 cases of T-PLL with flow cytometric evaluation of CD161 available. Six of these cases were positive for CD161 expression. All CD161-positive cases were positive for CD8 with variable CD4 expression, whereas all CD161-negative cases were negative for CD8. In a case with two neoplastic subsets positive and negative for CD8, only the former expressed CD161.ConclusionsThese novel results suggest that CD161 is often aberrantly expressed in a defined subset of T-PLL positive for CD8. We are showing the utility of this immunophenotype in diagnosis and follow-up.

scholarly journals Flow cytometric analysis of herpes simplex virus type 1 susceptibility to acyclovir, ganciclovir, and foscarnet.

1997 ◽  
Vol 41 (12) ◽  
pp. 2686-2692 ◽  
Author(s):  
I Pavić ◽  
A Hartmann ◽  
A Zimmermann ◽  
D Michel ◽  
W Hampl ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cytopathic Effect ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Resistant Strains ◽  
Simplex Virus

We established a quantitative flow cytometric method for determination of herpes simplex virus type 1 (HSV-1) susceptibility to acyclovir (ACV), ganciclovir, and foscarnet in vitro. Susceptibility was defined in terms of the drug concentration which reduced the number of cells expressing HSV-1 glycoprotein C (gpC) with a fluorescence intensity of > or =10(2) by 50% (IC50). Flow cytometry allowed us to use a high (1.0) as well as a low (0.005) multiplicity of infection, and determination of the IC50 was possible after one or more viral replicative cycles. IC50s were dependent on virus input and on time postinfection. In mixture experiments, 1 to 2% resistant viruses added to a sensitive strain could be detected. The results obtained by flow cytometry showed a good qualitative correlation with those achieved by cytopathic effect inhibitory assay. However, flow cytometry might detect more quantitative differences in drug susceptibility, especially among resistant strains, as confirmed also by determination of intracellular drug phosphorylation. The mean IC50s for ACV-sensitive strains were 0.45 to 1.47 microM, and those for ACV-resistant strains were between 140 and 3,134 microM. Flow cytometric analysis was fast and accurate, automatizable, and highly reproducible. Flow cytometry may be a more powerful tool than standard cytopathic effect-based assays and could have advantages for the detection of low levels of drug resistance or mixtures of sensitive and resistant virus strains.

176 Localization and Quantitative Expression of Phospholipase C Zeta in Equine Sperm Using Commercial Antibodies

2018 ◽  
Vol 30 (1) ◽  
pp. 228
Author(s):  
R. A. Gonzalez-Castro ◽  
J. K. Graham ◽  
E. M. Carnevale
Keyword(s):  
Flow Cytometry ◽  
Phospholipase C ◽  
Fluorescence Intensity ◽  
Molecular Probes ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Flow Cytometric ◽  
Live Sperm ◽  
Commercial Antibodies ◽  
Flow Cytometric Evaluation

Fertilization failure in vivo and in vitro (intracytoplasmic sperm injection, ICSI) can be caused by the inability of sperm to elicit intracellular calcium oscillations and to induce oocyte activation. Phospholipase C zeta (PLCz) is sperm-associated protein that can induce oocyte activation. Male infertility has been associated with PLCz deficiency in various species, although this has not been studied in the stallion. We hypothesised that the location and amount of PLCz on sperm varies among stallions. The aim of this study was to validate commercial antibodies (Ab) to detect PLCz on stallion sperm, and then to use these Ab to quantify the amount of PLCz, using flow cytometry, with the long-term goal of correlating PLCz on sperm with stallion fertility. Frozen-thawed sperm were analysed (20 stallions in 3 replicates) using 2 commercial Ab (anti-mouse PLCz M163 and anti-human PLCz H50, Santa Cruz Biotechnology, TX, USA). Western blot and immunofluorescence microscopy were used to validate Ab binding. For microscopy, sperm DNA was counterstained with 1 µg mL−1 Hoechst 33258. For flow cytometry, samples were incubated with Live Dead Fixable Far Red Stain Kit (Molecular Probes, Eugene, OR, USA), fixed, permeabilized, incubated overnight with primary Ab, and labelled with conjugated secondary Ab (anti-rabbit IgG Alexa Fluor 488, Molecular Probes). Green and far red mean fluorescence intensity (MFI) were measured for 20,000 cells per sample. Results are presented as mean ± SEM. Wilcoxon test, Spearman rank correlation, and linear regression were performed for analyses. Immunoblot analyses for both commercial Ab identified an immunoreactive band of ~70 kDa in sperm heads, tails, and whole sperm; β-tubulin was used as loading control and for normalization. Microscopy revealed PLCz in the acrosomal and post-acrosomal regions, connecting piece, midpiece, and tail. Post-acrosomal localization was the pattern most frequently observed (55%), followed by acrosomal plus post-acrosomal regions (25%). The PLCz labelling was observed on >85% of midpiece and tail regions, independent of Ab used. Flow cytometric evaluation revealed that percentage of live sperm was 47 ± 2%. Similar fluorescence intensity was exhibited for both Ab (M163 and H50) with a wide range of values among stallions [M163, mean 30.7 ± 1.9 × 103 (range, 8.8-82.2 × 103); H50: 25.5 ± 3.2 × 103 (7.3-55.0 × 103)]. The percentage of live sperm within a sample was not associated with Ab MFI. However, when samples were gated for live/dead cells, live sperm exhibited higher (P < 0.001) MFI than dead sperm for M163 (42.6 ± 6.0 v. 30.6 ± 3.9 × 103) and H50 (38.4 ± 4.7 v. 25.6 ± 3.7 × 103). There was a strong and positive correlation between M163 and H50 MFI for total sperm and live sperm (total: r = 0.81, P < 0.001; live: r = 0.71; P < 0.001). In conclusion, 2 anti-PLCz commercial antibodies detected equine PLCz, and the PLCz was localised on the sperm as described. Flow cytometric evaluation showed that stallions have different quantities of PLCz on their sperm, and this may provide a mean to determine if PLCz on stallion sperm is associated with fertility.

Flow cytometric evaluation of ki67 for the determination of malignancy grade in canine lymphoma

2013 ◽  
Vol 13 (4) ◽  
pp. 475-480 ◽  
Author(s):  
A. Poggi ◽  
B. Miniscalco ◽  
E. Morello ◽  
S. Comazzi ◽  
M. E. Gelain ◽  
...  
Keyword(s):  
Canine Lymphoma ◽  
Malignancy Grade ◽  
Flow Cytometric ◽  
Flow Cytometric Evaluation

scholarly journals Flow Cytometric Determination of Panton-Valentine Leucocidin S Component Binding

2001 ◽  
Vol 69 (4) ◽  
pp. 2390-2395 ◽  
Author(s):  
Valérie Gauduchon ◽  
Sandra Werner ◽  
Gilles Prévost ◽  
Henri Monteil ◽  
Didier A. Colin
Keyword(s):  
Flow Cytometry ◽  
Polymorphonuclear Neutrophils ◽  
Mutant Form ◽  
Binding Studies ◽  
Flow Cytometric ◽  
Kinase C ◽  
Specific Affinity ◽  
Substitution Mutant ◽  
Protein Kinase C Activation

ABSTRACT The binding of the S component (LukS-PV) from the bicomponent staphylococcal Panton-Valentine leucocidin to human polymorphonuclear neutrophils (PMNs) and monocytes was determined using flow cytometry and a single-cysteine substitution mutant of LukS-PV. The mutant was engineered by replacing a glycine at position 10 with a cysteine and was labeled with a fluorescein moiety. The biological activity of the mutant was identical to that of the native protein. It has been shown that LukS-PV has a high affinity for PMNs (K d = 0.07 ± 0.02 nM, n = 5) and monocytes (Kd = 0.020 ± 0.003 nM,n = 3) with maximal binding capacities of 197,000 and 80,000 LukS-PV molecules per cell, respectively. The nonspecifically bound molecules of LukS-PV do not form pores in the presence of the F component (LukF-PV) of leucocidin. LukS-PV and HlgC share the same receptor on PMNs, but the S components of other staphylococcal leukotoxins, HlgA, LukE, and LukM, do not compete with LukS-PV for its receptor. Extracellular Ca2+ at physiological concentrations (1 to 2 nM) has only a slight influence on the LukS-PV binding, in contrast to its complete inhibition by Zn2+. The down-regulation by phorbol 12-myristate 13-acetate (PMA) of the binding of LukS-PV was blocked by staurosporine, suggesting that the regulatory effect of PMA depends on protein kinase C activation. The labeled mutant form of LukS-PV has proved very useful for detailed binding studies of circulating white cells by flow cytometry. LukS-PV possesses a high specific affinity for a unique receptor on PMNs and monocytes.

Flow cytometric determination of DNA content in isolated nuclei of cereals

Genome ◽  
1988 ◽  
Vol 30 (4) ◽  
pp. 565-569 ◽  
Author(s):  
E. Hülgenhof ◽  
R. A. Weidhase ◽  
R. Schlegel ◽  
A. Tewes
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Internal Standard ◽  
Aegilops Speltoides ◽  
Isolated Nuclei ◽  
Triticum Timopheevi ◽  
Flow Cytometric ◽  
Nuclear Isolation ◽  
Cereal Plants

Isolated nuclei from cereal plants were used for quantitative determination of DNA per nucleus by flow cytometry. The technique is based on enhanced fluorescence of ethidium bromide and olivomycin when they bind to DNA. Nuclei were isolated from protoplasts derived from leaves of seedlings. The diploid Hordeum vulgare cv. Trumpf was used as an internal standard. Analysis of nuclei from several cultivars of hexaploid wheat (Triticum aestivum), Triticum durum var. hordeiforme, T. durum var. alexandrinum, Triticum araraticum, Triticum timopheevi, Triticum monococcum, Aegilops speltoides, Secale cereale, and one hexaploid and one octoploid triticale revealed significant intra- and inter-specific differences in DNA values per 2C nucleus. The advantages of the procedure are discussed along with its utilization for quantitative DNA measurement in Gramineae.Key words: nuclear isolation, flow cytometry, DNA content, cereals.

scholarly journals Determination of the Viability of Aeromonas hydrophila in Different Types of Water by Flow Cytometry, and Comparison with Classical Methods

2005 ◽  
Vol 71 (12) ◽  
pp. 7948-7954 ◽  
Author(s):  
Anna Pianetti ◽  
Tania Falcioni ◽  
Francesca Bruscolini ◽  
Luigia Sabatini ◽  
Elivio Sisti ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Aeromonas Hydrophila ◽  
Flow Cytometric Analysis ◽  
Staining Technique ◽  
Plate Count ◽  
Physiological Status ◽  
Traditional Methods ◽  
Flow Cytometric ◽  
Bacterial Content

ABSTRACT The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.

scholarly journals Flow Cytometric Determination of the Effects of Antibacterial Agents on Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. capricolum, and Mycoplasma mycoides subsp. mycoides Large Colony Type

2006 ◽  
Vol 50 (8) ◽  
pp. 2845-2849 ◽  
Author(s):  
Patricia Assunção ◽  
Nuno T. Antunes ◽  
Ruben S. Rosales ◽  
Carlos Poveda ◽  
Jose B. Poveda ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Antibacterial Agents ◽  
Mycoplasma Species ◽  
Colony Type ◽  
Flow Cytometric ◽  
Mycoplasma Capricolum ◽  
Large Colony ◽  
Mycoplasma Mycoides

ABSTRACT Flow cytometry together with SYBR green I and propidium iodide was used to study the effects of enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and tylosin on four mycoplasma species. Inhibition of mycoplasma growth could be detected by as early as 3 h after the start of treatment. The strongest effect was observed with enrofloxacin- and ciprofloxacin-treated cells.

scholarly journals Safe Determination of Susceptibility of Mycobacterium tuberculosis to Antimycobacterial Agents by Flow Cytometry

1999 ◽  
Vol 37 (3) ◽  
pp. 479-483 ◽  
Author(s):  
Andrea V. Moore ◽  
Scott M. Kirk ◽  
Steven M. Callister ◽  
Gerald H. Mazurek ◽  
Ronald F. Schell
Keyword(s):  
Flow Cytometry ◽  
Mycobacterium Tuberculosis ◽  
Flow Cytometric Analysis ◽  
Test Methods ◽  
Susceptibility Test ◽  
Flow Cytometric ◽  
Proportion Method ◽  
Bactec System ◽  
Drug Free

We showed previously that susceptibility testing forMycobacterium tuberculosis labeled with fluorescein diacetate could be accomplished rapidly by using flow cytometry. However, safety was a major concern because mycobacteria were not killed prior to flow cytometric analysis. In this study, we developed a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing M. tuberculosis organisms in drug-free and antimycobacterial agent-containing medium. The susceptibilities of 17 clinical isolates of M. tuberculosis to ethambutol, isoniazid, and rifampin were tested by the agar proportion and flow cytometric methods. Subsequently, all flow cytometric susceptibility test samples were inactivated by exposure to paraformaldehyde before analysis with a flow cytometer. Agreement between the results from the two methods was 98%. In addition, the flow cytometric results were available 72 h after the initiation of testing. The flow cytometric susceptibility assay is safe, simple to perform, and more rapid than conventional test methods, such as the BACTEC system and the proportion method.

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