An efficient culture method for generating large quantities of mature mouse splenic macrophages

Alatery, Attiya; Basta, Sameh

doi:10.1016/j.jim.2008.07.009

Cited by 54 publications

(75 citation statements)

References 47 publications

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“…The generated BMMs were CD11b + and F4/80 + (purity . 90%) (21). P2Y 6 -knockdown L929 cells were cultured in RPMI 1640 with 10% FBS and 2 mg/ml puromycin.…”

Section: Methodsmentioning

confidence: 99%

Extracellular UDP and P2Y6 Function as a Danger Signal To Protect Mice from Vesicular Stomatitis Virus Infection through an Increase in IFN-β Production

Tan

Yan

et al. 2014

The Journal of Immunology

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Extracellular nucleotides that constitute a “danger signal” play an important role in the regulation of immune responses. However, the function and mechanism of extracellular UDP and P2Y6 in antiviral immunity remain unknown. In this study, we demonstrated the in vitro and in vivo protection of UDP/P2Y6 signaling in vesicular stomatitis virus (VSV) infection. First, we demonstrated that VSV-infected cells secrete UDP from the cytoplasm as a danger signal to arouse surrounding cells. Meanwhile, expression of the UDP-specific receptor P2Y6 also was enhanced by VSV. Consequently, UDP protects RAW 264.7 cells, murine embryonic fibroblasts, bone marrow–derived macrophages, and L929 cells from VSV and GFP lentivirus infection. This protection can be blocked by the P2Y6 selective antagonist MRS2578 or IFN-α/β receptor–blocking Ab. VSV-induced cell death and virus replication were both enhanced significantly by knocking down and knocking out P2Y6 in different cells. Mechanistically, UDP facilitates IFN-β secretion through the p38/JNK- and ATF-2/c-Jun–signaling pathways, which are crucial in promoting antiviral immunity. Interestingly, UDP was released through a caspase-cleaved pannexin-1 channel in VSV-induced apoptotic cells and protected cells from infection through P2Y6 receptor in an autocrine or paracrine manner. Furthermore, UDP also protected mice from VSV infection through P2Y6 receptors in an acute neurotropic infection mouse model. Taken together, these results demonstrate the important role of extracellular UDP and P2Y6 as a danger signal in antiviral immune responses and suggest a potential therapeutic role for UDP/P2Y6 in preventing and controlling viral diseases.

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“…The generated BMMs were CD11b + and F4/80 + (purity . 90%) (21). P2Y 6 -knockdown L929 cells were cultured in RPMI 1640 with 10% FBS and 2 mg/ml puromycin.…”

Section: Methodsmentioning

confidence: 99%

Extracellular UDP and P2Y6 Function as a Danger Signal To Protect Mice from Vesicular Stomatitis Virus Infection through an Increase in IFN-β Production

Tan

Yan

et al. 2014

The Journal of Immunology

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“…42 Primary cultures of splenic macrophages were prepared as described, with slight modifications. 43 Spleens were perfused with PBS with Ca 2+ containing collagenase (100 U/mL) and DNAse (450 U/mL) (PBS-CaCD) (all from Sigma, St. Louis, MO, USA). Following perfusion, spleens were cut and incubated in PBS-CaCD for 45 min.…”

Section: T a S T O R T I F O U N D A T I O Nmentioning

confidence: 99%

“…at 37°C. The cells were then filtered, washed and cultured as previously described 43 at a concentration of 1¥10 6 cells/mL. Non-adherent cells were removed after 3 days.…”

Section: T a S T O R T I F O U N D A T I O Nmentioning

confidence: 99%

Physiologically aged red blood cells undergo erythrophagocytosis in vivo but not in vitro

Gottlieb¹,

Topaz²,

Cohen³

et al. 2012

Haematologica

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The online version of this article has a Supplementary Appendix. BackgroundThe lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes. Design and MethodsTo test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro. ResultsThe large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes. ConclusionsThe large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support. ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n

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“…BM from C57BL/6 mice were collected to generate BMderived Mø or BM-DC as described previously [35]. For BMderived Mø, after 3 days of culturing, the nonadherent cells were removed and fresh conditioned medium containing 20% of L929 supernatant was added.…”

Section: Preparation Of Bm-derived Mø and Bm-dcmentioning

confidence: 99%

“…The medium was changed 2 days later and the cells were tested after 5 days of culture. For BM-DC, cells were processed as described previously [35] and the medium (2 mL) was removed every 2 days and replaced with fresh medium. At day 6, the nonadherent cells were transferred into a new 6-well plate and left for 4 h before the loosely adherent cells (highly enriched CD11c 1 MHC-II 1 ) were harvested and used at this day in the assay.…”

Section: Preparation Of Bm-derived Mø and Bm-dcmentioning

confidence: 99%

The outcome of cross‐priming during virus infection is not directly linked to the ability of the antigen to be cross‐presented

et al. 2010

Self Cite

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The initiation of CD8 1 T cell (CTL) immune responses can occur via cross-priming. Recent data suggested a relationship between cross-presentation and immunodominance of epitope-specific T cells. To test this association, we evaluated the efficacy of crosspresentation for several virus epitopes in vitro and examined if this can be extrapolated in vivo. Employing lymphocytic choriomeningitis virus (LCMV), we demonstrate that the cross-presentation and cross-priming of LCMV antigens were dominated by NP396, but not NP205 when analyzing the LCMV-NP. Although with LCMV-GP, cross-presentation was dominated by GP276, and cross-priming was dominated by GP33. Importantly, although NP396 was significantly more efficient than GP33 in cross-presentation, cross-priming of their specific CTL was comparable. In a subsequent virus challenge after cross-priming, GP33-specific CTL dominated the response. Accordingly, based on our data, the ability of viral epitopes to be cross-presented in vitro does not entirely reflect what would occur in cross-priming. Thus, weak cross-presenting antigens may still cross-prime an efficient CTL response depending on other in vivo elements such as the naïve T-cell precursor frequencies.

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An efficient culture method for generating large quantities of mature mouse splenic macrophages

Cited by 54 publications

References 47 publications

Extracellular UDP and P2Y6 Function as a Danger Signal To Protect Mice from Vesicular Stomatitis Virus Infection through an Increase in IFN-β Production

Extracellular UDP and P2Y6 Function as a Danger Signal To Protect Mice from Vesicular Stomatitis Virus Infection through an Increase in IFN-β Production

Physiologically aged red blood cells undergo erythrophagocytosis in vivo but not in vitro

The outcome of cross‐priming during virus infection is not directly linked to the ability of the antigen to be cross‐presented

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