scholarly journals Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes

2018 ◽  
Vol 93 (7) ◽  
pp. 695-705
Author(s):  
Irina V. Khalo ◽  
Viktoriya S. Kozyreva ◽  
Roman V. Vakhrushev ◽  
Daria S. Patlai ◽  
Anna N. Shilova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Theoretical Consideration ◽  
Antibody Binding ◽  
Practical Implementation ◽  
Igg Antibody ◽  
Human Leukocytes ◽  
Quantitative Immunoassay ◽  
Calibration Free

Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages

2016 ◽  
Vol 89 (11) ◽  
pp. 1001-1009 ◽  
Author(s):  
Morten N. Andersen ◽  
Sinan N. H. Al-Karradi ◽  
Tue W. Kragstrup ◽  
Marianne Hokland
Keyword(s):  
Flow Cytometry ◽  
Fc Receptors ◽  
Antibody Binding ◽  
Human Monocytes ◽  
Monocytes And Macrophages ◽  
Human Monocytes And Macrophages

Phagocytosis of Bacteria by Human Leukocytes Measured by Flow Cytometry

1983 ◽  
Vol 174 (2) ◽  
pp. 182-186 ◽  
Author(s):  
C.-F. Bass e ◽  
O. D. Laerum ◽  
C. O. Solberg ◽  
B. Haneberg
Keyword(s):  
Flow Cytometry ◽  
Human Leukocytes

Use of Pacific Orange™ Dye with Qdot® Nanocrystals and Other Violet-Excited Dyes in Polychromatic Flow Cytometry.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3901-3901
Author(s):  
Gayle M. Buller ◽  
JiXiang Liu ◽  
Stephen Yue ◽  
Jolene A. Bradford ◽  
William L. Godfrey
Keyword(s):  
Flow Cytometry ◽  
Bandpass Filter ◽  
Live Cell ◽  
Blue Dye ◽  
Dead Cell ◽  
Band Pass Filter ◽  
Scatter Plot ◽  
Pass Filter ◽  
Human Leukocytes ◽  
Polychromatic Flow Cytometry

Abstract Violet-excited fluorochromes are becoming more commonly used in polychromatic flow cytometry experiments. However, violet-excited fluorochromes with emissions longer than 450 nm have been shown to produce small signals relative to the autofluorescent background, usable only on densely expressed antigens, and are sometimes excited by a 488 nm argon ion laser. We have developed a novel violet-excited organic fluor, Pacific Orange™ dye, which has an emission maximum at 551 nm and which is not excited by 488 nm light. Pacific Orange dye is at least twice as bright as the other green emitting violet excitable dyes, Cascade Yellow™ dye and Alexa Fluor® 430 dye. Pacific Orange dye (585/42 nm bandpass filter) can be used for two color immunophenotyping with Pacific Blue ™ dye (450/50 nm band pass filter) with minimal compensation. Data is shown comparing a human CD4/CD8 combination using a direct antibody conjugate with a Zenon® labeling reagent bound to a primary antibody. CD45 antigen is easily resolved with Pacific Orange dye, allowing CD45/SSC gating of leukocytes using violet excitation. Pacific Orange and Pacific Blue dyes can be paired with the violet-excited Fixable Aqua dead cell stain (525/50 nm bandpass filter) to exclude dead cells from immunofluorescence staining. (Figure 1) Finally, a five-color human peripheral blood leukocyte panel is shown using only violet excitation, and pairing Pacific Orange anti-CD8 and Pacific Blue anti-CD4 with Qdot® 605, Qdot 655, and Qdot 705 nanocrystal streptavidin conjugates used sequentially with biotinylated anti-CD19, anti-CD3, and anti-CD56. (Figure 2) Pacific Orange dye provides a tool to transfer detection of abundant target antigens from 488 nm excitation to the violet laser, enabling the use the 488 laser for another marker. In addition, the use of multiple violet-excited dyes can enable the detectection of five or more additional markers to standard laser combinations for greater multiplexing in polychromatic flow cytometry. Figure 1. Immunophenotyping of mixed live and heat-killed human leukocytes using Pacific Orange dye, Pacific Blue dye and the Fixable Aqua dead cell reagent. Live cell events (Fixable Aqua stain-negative) were gated in the histogram (left) for display in the CD4/CD8 scatter plot (right). Figure 1. Immunophenotyping of mixed live and heat-killed human leukocytes using Pacific Orange dye, Pacific Blue dye and the Fixable Aqua dead cell reagent. Live cell events (Fixable Aqua stain-negative) were gated in the histogram (left) for display in the CD4/CD8 scatter plot (right). Figure 2. Five-color immunophenotyping of human leukocytes with Pacific Orange dye, Pacific Blue dye and three Qdot nanocrystal streptavidin conjugates using violet excitation. The Qdot nanocrystal staining was done with sequential staining and washing with biotinylated primary antibodies and streptavidin conjugates. Figure 2. Five-color immunophenotyping of human leukocytes with Pacific Orange dye, Pacific Blue dye and three Qdot nanocrystal streptavidin conjugates using violet excitation. The Qdot nanocrystal staining was done with sequential staining and washing with biotinylated primary antibodies and streptavidin conjugates.

Study of phagocytosis of Staphylococcus aureus and latex microbeads by human leukocytes using flow cytometry

1992 ◽  
Vol 76 (2) ◽  
pp. 259-259
Author(s):  
Rabesandratana Herisao ◽  
Chateau Marie-Thérèse ◽  
Caravano René ◽  
Serre Arlette
Keyword(s):  
Staphylococcus Aureus ◽  
Flow Cytometry ◽  
Human Leukocytes

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.

scholarly journals Cross-Strain Platelet Immunization in Selected Mouse Strains Recapitulates Clinical and Serologic Findings Typical of Post-Transfusion Purpura (PTP)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2349-2349
Author(s):  
Daniel W. Bougie ◽  
Jessica Sutton ◽  
Richard H. Aster
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Amino Acid ◽  
Conflicts Of Interest ◽  
Human Condition ◽  
Autoimmune Response ◽  
Single Amino Acid ◽  
Mouse Strains ◽  
Igg Antibody ◽  
Platelet Counts

Post-transfusion purpura (PTP) is an uncommon but life-threatening condition characterized by profound thrombocytopenia (TP) occurring one week after transfusion of blood products. The hallmark of PTP is a potent IgG antibody specific for a transfused human platelet antigen (HPA), usually HPA-1a located on αIIb/β3 integrin (GPIIb/IIIa). It is widely thought that, in PTP, the alloantibody somehow causes destruction of the recipient's platelets even though they lack the antigen for which the alloantibody is specific. Several reports have suggested that the underlying cause of PTP is a platelet-specific autoantibody that can be difficult to detect because it is absorbed in the process of destroying autologous platelets and is overshadowed by the accompanying, very potent alloantibody but experimental support for this concept is minimal. Platelet alloantigens comparable to HPAs have not been defined in animals. Using a public database, we identified four mouse strains (C57BL/6J (C57), 129S1/Svlmj (129), PWK/PhJ (PWK), AND SPRET/EIJ) differing from each other at amino acid residues in extracellular domains of GPIIb/IIIa that could comprise potential alloantigens. Cross-strain platelet immunizations (intraperitoneal with adjuvant) were performed weekly for 5 weeks while monitoring platelet counts and platelet associated IgG (PAIgG) and saving plasma samples for serologic studies. After 2-4 immunizations, each of 39 cross-strain but none of 9 strain-identical immunizations induced "alloantibodies" that recognized donor but not recipient platelets (flow cytometry). Thrombocytopenia (<50% of maximum platelet count) developed in 28 of 39 mice (71%) given strain-disparate platelets but not in mice given strain-identical platelets; 12 of these mice (30%) developed profound TP (<15%). The most consistent and severe declines in platelet counts occurred in PWK mice immunized with 129 platelets and vice versa (N=13) in which the mean platelet count decline was 88% (range 59-96%, median 92%). Autoantibodies recognizing syngeneic platelets were identified in all animals that developed profound TP and their potency (measured by flow cytometry) correlated closely with the severity of TP (p<0.001) (Fig 1). Alloantibodies were shown by immunoprecipitation to be mainly specific for GPIIb/IIIa (N=13) and GPIb/IX (N=1) on donor platelets. Two monoclonal antibodies (mAbs MBC417.1 and MBC425.1) specific for a single polymorphic amino acid at positions 111(Gly) and 37(Val), respectively, on GPIIb of C57 and PWK mice were generated using spleen cells of two immunized mice. To our knowledge, these are the first alloantibodies in mice that are specific for single amino acid polymorphisms in a platelet membrane glycoprotein and are thus comparable to HPA antibodies found in humans. The findings define a model (platelet immunization between PWK and 129 mice) in which a routine alloantibody response recognizing GPIIb/IIIa on donor platelets regularly transitions to an autoimmune response capable of causing profound thrombocytopenia, thus mimicking the course of PTP in human patients and supporting the hypothesis that PTP is an autoimmune disorder. Successful development of the model could be related to use of the more recently developed, wild-caught PWK strain as one of the partners for immunization. The inherent utility of a mouse model is expected to facilitate further work to define the molecular basis for a transition from allo- to auto-immunity in the human condition, post-transfusion purpura. Disclosures No relevant conflicts of interest to declare.

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