Flow Cytometry for the Isolation and Characterization of Rodent Meiocytes

Abstract Neonatal alloimmune thrombocytopenia (NAIT) can cause severe complications such as intrauterine death or intracranial hemorrhage (ICH) in the newborn, and is caused by the transfer of platelet-depleting antibodies from the mother to the fetus during pregnancy. These antibodies react with allogeneic epitopes, most commonly human platelet antigen (HPA) 1a, when present on fetal platelets. Although these responses are thought to be a result of a T cell-dependent immune response, HPA 1a specific T cells have not yet been isolated. To examine whether HPA 1a specific T cells could be detected and isolated, we collected PBMC post delivery from an HPA 1a negative mother who gave birth to an HPA 1a positive neonate suffering from severe thrombocytopenia (platelet count <50×109/L). The cells were stimulated with HPA 1a peptides (20aa) in long term cultures supplemented with IL-7 and IL-2, and subsequently, IL-15. After 4 weeks in culture these cells were labeled with CFSE dye and restimulated with HPA 1a or control peptides. After additional 2 weeks in culture supplemented with IL-2 and IL-15, specific proliferative responses were detectable by CFSE dye dilution by flow cytometry. The cells were cloned by fluorescent-activated cell sorting (FACS) and expanded in numbers with anti-CD3 stimulation in the presence of irradiated allogeneic PBMC and IL-2. The resulting clonal T cell lines were characterized in proliferation assays, ELISPOT assays and phenotyped by flow cytometry. All clones were CD3+, CD4+ and CD19−, and the majority of the clones proliferated and secreted cytokines in response to stimulation with HPA 1a peptides, but not control peptides. In ELISPOT assays, peptide-pulsed antigen-presenting cells were required for T cell detection. These clonal HPA 1a specific CD4+ T cell lines represent formal evidence of the existence of HPA 1a specific T cell responses related to NAIT and will serve as important tools for further characterization of maternal immune responses associated with NAIT.

Download Full-text

A Flow Cytometry-Based Approach for the Isolation and Characterization of Neural Stem Cell Primary Cilia

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2018.00519 ◽

2019 ◽

Vol 12 ◽

Cited By ~ 1

Author(s):

Sara Monaco ◽

Katja Baur ◽

Andrea Hellwig ◽

Gabriele Hölzl-Wenig ◽

Claudia Mandl ◽

...

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

Neural Stem Cell ◽

Primary Cilia ◽

Isolation And Characterization

Download Full-text

Isolation and Characterization of Cell- and Size-Specific Extracellular Vesicles by Nanoscale High Resolution Flow Cytometry

Microscopy and Microanalysis ◽

10.1017/s1431927619006317 ◽

2019 ◽

Vol 25 (S2) ◽

pp. 1116-1117

Author(s):

Terry K. Morgan

Keyword(s):

Flow Cytometry ◽

High Resolution ◽

Extracellular Vesicles ◽

Isolation And Characterization

Download Full-text

Isolation and Characterization of Antigen-Specific Plasmablasts Using a Novel Flow Cytometry–Based Ig Capture Assay

The Journal of Immunology ◽

10.4049/jimmunol.1701253 ◽

2017 ◽

Vol 199 (12) ◽

pp. 4180-4188 ◽

Cited By ~ 10

Author(s):

Christopher L. Pinder ◽

Sven Kratochvil ◽

Deniz Cizmeci ◽

Luke Muir ◽

Yanping Guo ◽

...

Keyword(s):

Flow Cytometry ◽

Capture Assay ◽

Isolation And Characterization

Download Full-text

POLYCLONAL ANTIBODIES AGAINST HUMAN PLASMINOGEN: PURIFICATION, CHARACTERIZATION AND APPLICATION

Biotechnologia Acta ◽

10.15407/biotech13.06.050 ◽

2020 ◽

Vol 13 (6) ◽

pp. 50-57

Author(s):

T. A. Yatsenko ◽

Keyword(s):

Flow Cytometry ◽

Polyclonal Antibodies ◽

Blood Collection ◽

Salting Out ◽

Cell Functions ◽

Isolation And Characterization ◽

Wide Range ◽

Direct Elisa ◽

Human Plasminogen

The plasminogen/plasmin system plays a crucial role in fibrinolysis and regulation of cell functions in a wide range of normal and pathological processes. Investigation of plasminogen/plasmin functions requires the availability of well-characterized and effective molecular tools, such as antibodies. In the present work, the isolation and characterization of rabbit polyclonal antibodies against human plasminogen are described and approaches for the identification of plasminogen and its fragments using the purified antibodies are demonstrated. For the antibodies isolation, standard animal immunization and blood collection procedures, serum isolation, protein salting out and affinity chromatography were performed. For the antibodies characterization and application, the following methods were used: enzyme linked immunoassay (ELISA), Western blotting, FITC-protein conjugation, flow cytometry and spectrofluorometry. The obtained polyclonal rabbit anti-human plasminogen antibodies interacted with human Glu- and Lys-plasminogen, kringles 1-3 and 1-4 of plasminogen, mini-plasminogen, the heavy and light chain of plasmin. We propose the application of anti-plasminogen antibodies for the direct ELISA, Western blot analysis, and for flow cytometry and spectrofluorometric analysis of plasminogen binding with cells. The obtained anti-plasminogen antibodies are promising tools for the investigation of plasminogen/plasmin system functions, either fibrinolytic or signaling.

Download Full-text