Protocol for Adhesion and Immunostaining of Lymphocytes and Other Non-Adherent Cells in Culture

Tsang, Matthew; Gantchev, Jennifer; Ghazawi, Feras M.; Litvinov, Ivan V.

doi:10.2144/000114610

Cited by 49 publications

(34 citation statements)

References 10 publications

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“…Indirect immunofluorescence was performed by adhering and staining lymphocytes onto 8-well chamber slides (Thermo Fisher) coated with Poly-L-Lysine (Sigma-Aldrich) as previously described in our protocol [54]. A Zeiss LSM 510 confocal microscope (Zeiss, Oberkochen, Germany) was used for all cell imaging.…”

Section: Methodsmentioning

confidence: 99%

A study of meiomitosis and novel pathways of genomic instability in cutaneous T-cell lymphomas (CTCL)

et al. 2018

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Genomic instability is a hallmark of cancer and an enabling factor for genetic alterations that drive cancer development and progression. The clashing of mitosis and aberrantly expressed meiosis machineries, which may contribute to genomic instability, has been coined cancer “meiomitosis”. LINE-1 retrotransposition, a process active in germ cells, acts outside of the meiotic machinery to create DNA double strand breaks (DNA DSBs) and has played an important role in the evolution of the human genome. We have previously demonstrated that in CTCL several cancer testis/meiotic genes are expressed. Furthermore, this cancer exhibits extensive and ongoing chromosomal/microsatellite instability. In this study we analyzed immortalized patient-derived cells and primary CTCL patient samples using RT-PCR, western blotting and confocal microscopy and found that proteins critically involved in meiosis and LINE-1 retrotransposition are expressed and are associated with chromosomal instability and DNA DSB formation. Using cell cycle synchronization, we show G1/S phase-transition-specific expression of meiosis proteins. Using the Alu retrotransposition assay, we demonstrate the functional activity of LINE-1 retrotransposon in CTCL. Histone acetyltransferase inhibition results in downregulation of the ectopic germ cell programs and concomitant decrease in DNA DSBs foci formation. Notably, LINE-1 and meiosis genes were expressed across a panel of other solid tumor cell lines. Taken together, our results indicate that malignant cells in culture undergo “cancer meiomitosis” rather than the classic mitosis division. The ectopic expression of meiosis genes and reactivation of LINE-1 may be contributing to genomic instability and represent novel targets for immunotherapy in this and other cancers.

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Section: Methodsmentioning

confidence: 99%

A study of meiomitosis and novel pathways of genomic instability in cutaneous T-cell lymphomas (CTCL)

et al. 2018

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“…Vero cells were seeded on 13 mm diameter coverslips (Knittel-Glass, Braunschweig, Germany) in 24-well plates (2 × 10 4 cells/mL) and infected with OROV. Nonadherent cells such as leukocytes lineages and human PBMC were seeded in 8 wells Lab-Tek ® Glass Chamber Slide™ (Thermo Fisher Scientific, Waltham, MA, USA) in PBS 1x and incubated at 37 • C for 30 min [34]. Then, PBS was removed for the addition of media with virus inoculum.…”

Section: Orov Proteins Genome and Antigenome Detection By Confocal Mmentioning

confidence: 99%

Oropouche Virus Infects, Persists and Induces IFN Response in Human Peripheral Blood Mononuclear Cells as Identified by RNA PrimeFlow™ and qRT-PCR Assays

Amorim

Pontelli

Souza

et al. 2020

Viruses

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Oropouche orthobunyavirus (OROV) is an emerging arbovirus with a high potential of dissemination in America. Little is known about the role of peripheral blood mononuclear cells (PBMC) response during OROV infection in humans. Thus, to evaluate human leukocytes susceptibility, permissiveness and immune response during OROV infection, we applied RNA hybridization, qRT-PCR and cell-based assays to quantify viral antigens, genome, antigenome and gene expression in different cells. First, we observed OROV replication in human leukocytes lineages as THP-1 monocytes, Jeko-1 B cells and Jurkat T cells. Interestingly, cell viability and viral particle detection are maintained in these cells, even after successive passages. PBMCs from healthy donors were susceptible but the infection was not productive, since neither antigenome nor infectious particle was found in the supernatant of infected PBMCs. In fact, only viral antigens and small quantities of OROV genome were detected at 24 hpi in lymphocytes, monocytes and CD11c+ cells. Finally, activation of the Interferon (IFN) response was essential to restrict OROV replication in human PBMCs. Increased expression of type I/III IFNs, ISGs and inflammatory cytokines was detected in the first 24 hpi and viral replication was re-established after blocking IFNAR or treating cells with glucocorticoid. Thus, in short, our results show OROV is able to infect and remain in low titers in human T cells, monocytes, DCs and B cells as a consequence of an effective IFN response after infection, indicating the possibility of leukocytes serving as a trojan horse in specific microenvironments during immunosuppression.

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“…PBMCs were then re-suspended in serum free RPMI 1640, L-glutamine supplemented medium (Gibco Lifetechnologies, Carlsbad, CA, USA) and plated on glass coverslips at a density of (2 × 10 6 ) per condition. Cells were allowed to adhere to the glass surfaces for 2 h at 37 • C and 5% CO 2 [33] before experiments.…”

Section: Human Blood Pbmcs Isolationmentioning

confidence: 99%

A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

Tumpara

Korenbaum

Kühnel

et al. 2021

IJMS

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The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

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Protocol for Adhesion and Immunostaining of Lymphocytes and Other Non-Adherent Cells in Culture

Cited by 49 publications

References 10 publications

A study of meiomitosis and novel pathways of genomic instability in cutaneous T-cell lymphomas (CTCL)

A study of meiomitosis and novel pathways of genomic instability in cutaneous T-cell lymphomas (CTCL)

Oropouche Virus Infects, Persists and Induces IFN Response in Human Peripheral Blood Mononuclear Cells as Identified by RNA PrimeFlow™ and qRT-PCR Assays

A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

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