Fluorescence intensity and lifetime measurement of free and particle-bound fluorophore in a sample stream by phase-sensitive flow cytometry

1999 ◽  
Vol 70 (12) ◽  
pp. 4682-4688 ◽  
Author(s):  
John A. Steinkamp ◽  
Jan F. Keij
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Lifetime Measurement ◽  
Sample Stream ◽  
Phase Sensitive

scholarly journals A flow cytometric assay of neutrophil degranulation.

1983 ◽  
Vol 31 (6) ◽  
pp. 737-744 ◽  
Author(s):  
W R Abrams ◽  
L W Diamond ◽  
A B Kane
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Ionophore A23187 ◽  
Flow Cytometric Assay ◽  
Elastase Activity ◽  
Flow Cytometric ◽  
Specific Granules ◽  
Neutrophil Degranulation ◽  
High Fluorescence Intensity ◽  
High Fluorescence

A quantitative assay of neutrophil degranulation was developed using flow cytometry. Dog neutrophils were purified to greater than 95% purity and viability by isopyknic density centrifugation in an isosmotic medium. These cells concentrated the fluorochrome acridine orange (AO) in their azurophilic granules, but not in specific granules. Also contained in the azurophilic granules are elastase, myeloperoxidase, and approximately 50% of the lysozyme activity. The fluorochrome was released concomitantly with elastase activity, as shown by flow cytometry, fluorescence microscopy, and biochemical assay in response to the ionophore A23187. By flow cytometry, unstimulated cells are distributed in a single broad peak of high fluorescence intensity. With increasing concentrations of A23187 (0.48-4.80 microM), a greater proportion of the cells shifted to a single peak of low fluorescence intensity. Few cells with intermediate fluorescence were observed. These analyses revealed that the neutrophils degranulated in a quantal, all-or-none response.

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.

Megakaryocyte-Active Chemokines: Dysregulation in the SDF-1a/CXCR4 Axis in Patients with Essential Thrombocythemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4969-4969
Author(s):  
Juan P. Salim ◽  
Rosana F. Marta ◽  
Felisa C. Molinas
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Essential Thrombocythemia ◽  
Plasma Levels ◽  
Platelet Membrane ◽  
Rank Test ◽  
Control Group ◽  
Expression Levels ◽  
Normal Controls ◽  
Cxcr4 Axis

Abstract Chemokines belong to a large family of molecules that are implicated in the localization and production of blood cells. Some of them, such as Interleukin 8 (IL-8) and GRO-a, participate in the regulation of megakaryopoiesis, mostly by exerting an inhibitory action. Recently, the involvement of stromal derived factor 1 (SDF-1) in synergizing the stimulatory effect of thrombopoietin on megakaryopoiesis and its participation in megakaryocyte transendothelial migration has been described. The aim of the present study was the evaluation of the plasma levels of IL-8, GRO- a and SDF-1 in patients with essential thrombocythemia (ET), a myeloproliferative disorder characterized by megakaryocytic hyperproliferation with increased circulating platelet count. Besides, the corresponding chemokine receptors were assayed on platelet membrane from ET patients. A cohort of 27 patients diagnosed according to the Polycitemia Vera Study Group were enrolled in the study (mean age, 45; 21 women). Twenty-seven normal subjects matched by sex and age were taken as the control group. The Ethic Committee from IDIM A. Lanari approved the study and all patients and normal controls signed the informed consent. Plasma levels of the chemokines were measured by ELISA technique (R&D Systems) according to the manufacturer. Expression levels of IL-8 receptors (CXCR1 and CXCR2), GRO-a receptors (CXCR2) and SDF-1 receptors (CXCR4) on platelet membrane were evaluated by flow cytometry using specific MoAbs and the corresponding isotype controls (B-D Pharmingen). Flow cytometry results were expressed as relative fluorescence intensity (RFI, the relationship between the mean fluorescence intensity from the specific antibody and the isotype control). Results were expressed as median and range. Statistic analysis was carried out using Mann-Whitney Wilcoxon rank sum test and Wilcoxon signed rank test. Plasma levels of the chemokines measured in 19 ET patients were similar to that found in normal controls, IL-8 2.5, pg/ml (0.8–28.2) and 2.8 pg/ml (1.1–16.5), GRO-a, 30.0 pg/ml (7.4–463.1) and 23.9 pg/ml (9.6–148.0), SDF-1, 1895.0 pg/ml (1246.0–2719.0) and 1915.0 pg/ml (822–2424.0), respectively. The expression levels of CXCR4 receptor was found diminished in platelets from ET patients, RIF 16.94 (1.3–31.3) compared to normal controls, 27.4 (2.4–58.4); p=0.0059, n=10. However, the expression of CXCR1 and CXCR2 in platelets from ET patients was normal. In conclusion, although plasma levels of the chemoquines IL-8, GRO-a and SDF-1 were normal in these patients, the decreased level of CXCR4 on platelet membrane suggests a dysregulation in the SDF-1a/CXCR4 axis in patients with essential thrombocythemia.

Flow Cytometry Thresholds of Myeloperoxidase Detection to Discriminate Between Acute Lymphoblastic or Myeloblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1450-1450 ◽  
Author(s):  
Julien Guy ◽  
Iléana Antony-Debré ◽  
Isabelle Arnoux ◽  
Chantal Fossat ◽  
Emmanuel Benayoun ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Fluorescence Intensity ◽  
Conflicts Of Interest ◽  
Specific Binding ◽  
Negative Control ◽  
Analysis Method ◽  
Isotype Control ◽  
Blast Cells ◽  
Negative Controls

Abstract Abstract 1450 Background: The WHO 2008 classification emphasizes the role of myeloperoxidase (MPO) detection as the only requirement for assigning myeloid lineage to a blast population, notably for the diagnosis of acute leukemia of ambiguous lineage. WHO highlights flow cytometry (FCM) as the preferred method for MPO detection and EGIL proposed a 10% cut-off for FCM but 3% for cytochemistry. Here we performed a reevaluation of the MPO positivity threshold by FCM comparing as background reference isotype controls or the autofluorescence of residual normal lymphocytes. Methods: A multicenter retrospective trial was set-up which compared retrospectively 128 acute lymphoblastic leukemias (ALL) and 75 acute myeloid leukemias without maturation (AML1), defined as MPO negative or positive by benzidine-based cytochemistry. Blasts were considered MPO positive by flow cytometry when their mean fluorescence intensity exceeded that of blast cells incubated with an isotype control or that of residual lymphocytes in the MPO-stained sample. In order to homogenize the interpretation of MPO staining, five various cut-offs were assessed for both negative controls, respectively 2%, 1%, 0.75%, 0.5% and 0.25%. Besides, as Ratio Fluorescence Intensity (RFI) may be a useful parameter to analyze staining for markers with unimodal distribution, we evaluated the RFI of blast cells MPO fluorescence relative to both controls, comparing mean and median intensities. Results: The harmonized method that was developed to interpret MPO staining by FCM between 4 French centers can be applied regardless of antibodies, permeabilization reagents or instruments used. For both negative controls, the 1% cut-off provided the best discrimination by ROC curve, and was used to assess the percentage of stained blasts in the 203 cases. The EGIL 10% threshold of stained blasts to discriminate between ALL and AML, using the isotype control to assess positivity, provided 100% sensitivity and 85.4% specificity, the optimal threshold being 13% (sensitivity 95.1%, specificity 91.7%). Residual normal lymphocytes proved to be an advantageous alternative reference, a threshold of 28% yielding improved 97.4% sensitivity and 96.1% specificity (Figure). The correlation between both methods was excellent, yet the percentage of positive blasts was higher using lymphocytes as control compared to isotype control. Using the RFI method, the isotype control appeared as the more relevant negative control to discriminate ALL against AML1 with a threshold of 3.42. However, with 90% sensivity and 95,83% specificity, the RFI method was less performing than percentages in this study. Finally, we assessed the relevance of this analysis method and positivity thresholds on 18 AMLs with minimal differentiation (AML0), MPO-negative by definition in cytochemistry, yet liable to be positive in FCM. Interestingly, with these new appropriate thresholds, MPO staining was positive for 10 of 18 AML0 when using lymphocytes as negative controls and only 3 of 17 cases when using an isotype control. Conclusion: i) MPO detection by flow cytometry can be interpreted indifferently of the negative control used if an appropriate threshold is applied; ii) Our analysis method of MPO expression is relevant for ALL and AML discrimination and can be useful for AML0 or mixed phenotype acute leukemia assessment, especially using residual lymphocytes as reference, since the latter bypass the higher non-specific binding of isotype controls on blast cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro observation: the GFP-E. coli adhering to porcine erythrocytes can be removed by porcine alveolar macrophages

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6439 ◽  
Author(s):  
Wei Yin ◽  
Chun Wang ◽  
Kuohai Fan ◽  
Na Sun ◽  
Yaogui Sun ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Fluorescence Intensity ◽  
Alveolar Macrophages ◽  
Control Group ◽  
Laser Confocal Microscopy ◽  
Functional Protein ◽  
Blank Control Group ◽  
E Coli

Although the activation of pathogen phagocytosis via complement system has been studied, erythrocyte-phagocyte interactions in pigs are not clearly understood. Therefore, we sought to investigate the ability of porcine erythrocytes to clear immune complexes (ICs) by using laser confocal microscopy and flow cytometry to observe the immune adhesion of porcine erythrocytes to fluorescent bacilli and the immune presentation process of transferring fluorescent bacilli to macrophages. Isolated porcine alveolar macrophages (PAMs) had uniform morphology and size, and a survival rate of 97.2%. The phagocytosis rate was 98.8%. After WT E. coli was labeled with Fluorescein Isothiocyanate (FITC), the bacteria showed a bright green fluorescence, and the labeling rate was 92.3%. When laser confocal microscopy was utilized to observe the co-incubation system of porcine erythrocytes, PAM, and fluorescent E. coli, the fluorescence intensity of bacilli decreased with increasing observation time and even disappeared. Flow Cytometry examination showed that the average fluorescence intensity of PAMs co-incubated with porcine erythrocytes adhered to WT-E. coli-FITC, was significantly higher than that of normal PAMs. Furthermore, when porcine erythrocytes adhered to WT E. coli were incubated with PAMs, the surface mean fluorescence intensity of porcine erythrocytes was significantly higher than that of the blank control group. This shows that PAMs can competitively bind to the oposinized E. coli adhered to the surface of porcine erythrocytes, and these oposinized pathogens can enter macrophages by the process of phagocytosis, which promoting the internalization of ICs or pathogens. During this process, the physical morphology of porcine erythrocytes was not damaged, but the levels of its main functional protein CR1-like were reduced.

scholarly journals Incorporation of stilbazolium merocyanines into human leukocytes measured by flow cytometry.

1995 ◽  
Vol 42 (3) ◽  
pp. 333-338 ◽  
Author(s):  
K Wiktorowicz ◽  
M Niedbalska ◽  
A Planner ◽  
D Frackowiak
Keyword(s):  
Flow Cytometry ◽  
Cell Membrane ◽  
Fluorescence Intensity ◽  
Peripheral Blood ◽  
Incubation Temperature ◽  
Human Peripheral Blood ◽  
Dye Molecules ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Human Leukocytes

Human peripheral blood leukocytes were incubated with thirteen various merocyanines of the stilbazolium betaine type and the fluorescence intensities of the cells were measured by flow cytometry. The fluorescence intensity of lymphocytes, monocytes and granulocytes depended on the time and temperature of incubation with the dyes. An increase in the incubation temperature enhanced the fluorescence intensity whereas washing of the cells after incubation had little influence on the observed emission. This points to incorporation of the dye molecules into the cell membrane. From the measured fluorescence intensities corrected for relative fluorescence yields, the relative efficiencies of incorporation into the cells of the various merocyanines tested were evaluated. The efficiency was dependent on the type of the cells and the lenght and side groups of the merocyanine molecules studied.

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