Simultaneous blockade of Fcγ receptors and indirect labeling of mouse lymphocytes by the selective detection of allotype‐restricted epitopes on the kappa chain of rat monoclonal antibodies

Balogh, Péter; Tew, John G.; Szakal, Andras K.

doi:10.1002/cyto.10057

Cited by 4 publications

(3 citation statements)

References 6 publications

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“…Collectively, we were targeting the removal of monocytes, as well as glial, amacrine and photoreceptor cells. Prior to cell surface labeling, we added purified mouse anti-CD16/32 antibody to block FcγRII/III, thus reducing false positive immunoreactivity (Unkeless et al, 1979 ; Balogh et al, 2002 ). Our flow cytometry-based cell sorting validation studies included examination of pre- (Figure 3B ) and post-sorted cells (Figure 3C ) to confirm that the post-sorted cells that were isolated using cell surface markers expressed all four RGC intracellular markers: SNCG, BRN3A, TUJ1, and RBPMS.…”

Section: Resultsmentioning

confidence: 99%

Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

Chintalapudi

Djenderedjian

Stiemke

et al. 2016

Front. Aging Neurosci.

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Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

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Section: Resultsmentioning

confidence: 99%

Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

Chintalapudi

Djenderedjian

Stiemke

et al. 2016

Front. Aging Neurosci.

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“…Bone marrow in Giemsa stained sections from the head of the tibia was evaluated for estimation of myeloid:erythroid (M:E) ratio. (23). Following Fc block, cells were incubated for 25 min with panels of rat anti-murine monoclonal antibodies to lineage phenotypic markers.…”

Section: Histological Analysismentioning

confidence: 99%

Myelodysplasia and anemia of chronic disease in human tumor necrosis factor‐α transgenic mice

Capocasale¹,

Makropoulos²,

Achuthanandam³

et al. 2008

Cytometry Pt A

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TNF-a is a pleitropic cytokine that expresses both pro-and anti-inflammatory activity and transgenic mice expressing human tumor necrosis factor-a (TNF-a) exhibit a progressive polyarthritis that models rheumatoid arthritis (RA). One of the common comorbidities of RA is anemia of chronic disease (ACD). The purpose of these experiments was to study the changes in the bone marrow and peripheral blood that accompany polyarthritis in TNF-a transgenic mice in an effort to better understand the pathogenesis of myelodysplasia and ACD. Polychromatic cytometry, hematology and serum cytokine analysis were used to study the pathogenesis of ACD in human TNF-a transgenic mice. Our hematological evaluation revealed a mild, compensated, microcytic hypochromic anemia, and monocytosis. In the bone marrow, we observed alterations in cell kinetics, decreased relative expression of transferrin receptor and increased apoptosis and cell death in several late precursor cell populations. Although significant levels of human TNF-a were found in the serum, neither change in serum murine erythropoietin nor any significant difference observed in serum levels of murine IL-b, IL-5, IL-6, IL-10, IL-12(p70), IL-17, TNF-a, IFNg, GM-CSF, MIP-1aJE, MCP-5 was observed. Tg197 mice develop a compensated, microcytic, hypochromic anemia, and a functional iron deficiency by 9 weeks of age. Changes in peripheral blood are reflected in alterations in cell kinetics, transferrin receptor expression and markedly increased apoptosis and cell death in the bone marrow indicating that TNF-a may contribute to myelodysplasia in ACD. Moreover, since human TNF-a can interact only with murine TNFR1, our data suggest that TNFR1 may play an important role in the development of ACD. '

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“…Collectively, we were targeting the removal of monocytes, as well as glial, amacrine and photoreceptor cells. Prior to cell surface labeling, we added purified mouse anti-CD16/32 antibody to block FcRII/III, thus reducing false positive immunoreactivity (224) (224,225). Our flow cytometry-based cell sorting validation studies included examination of pre- (Figure 4-9) and post-sorted cells (Figure 4-10) to confirm that the post-sorted cells that were isolated using cell surface markers expressed all four RGC intracellular markers: SNCG, BRN3A, and TUJ1, and RBPMS.…”

Section: Confirmation Of Additional Surface Markers To Be Used As Negmentioning

confidence: 99%

Multipronged Approach to Study Glaucoma-Associated Phenotypes

Chintalapudi¹

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AndMy better half, Purav Trivedi iv ACKNOWLEDGEMENTSFirst of all, I wish to thank the Almighty Lord for helping me start, pursue and successfully complete my PhD. Working as PhD student in UTHSC was a magnificent as well as challenging experience for me. This dissertation was the product of a large measure of serendipity, fortuitous encounters with people who have changed the course of my academic career. It would not have been possible without the help of so many people in so many ways. Here is a tribute to all those people.

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Simultaneous blockade of Fcγ receptors and indirect labeling of mouse lymphocytes by the selective detection of allotype‐restricted epitopes on the kappa chain of rat monoclonal antibodies

Cited by 4 publications

References 6 publications

Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

Myelodysplasia and anemia of chronic disease in human tumor necrosis factor‐α transgenic mice

Multipronged Approach to Study Glaucoma-Associated Phenotypes

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