To generate bone marrow-derived macrophages, mouse bone marrow monocytes were isolated from C C57BL/6J and TLR2−/− (B6.129-Tlr2tm1Kir/J) mice (Jackson Laboratories) and cultured in L929 medium, a source of macrophage colony-stimulating factor, for 7 days 49 (link). Human primary peripheral blood mononuclear cells (PBMC; Precision for Medicine, Frederick, MD) were cultured in RPMI-1640 media supplemented with 20ng/mL of recombinant human M-CSF. THP-1 human monocytic cells (ATCC) were differentiated with 5 ng/ml of phorbol 12-myristate 13-acetate as described50 . Cells were infected with RV-A1B and RV-C15 at a multiplicity of infection (MOI) of 1.0 at 33°C. To study virus entry, human THP-1-derived macrophages and mouse bone marrow-derived macrophages were incubated with RV-A1B or RV-C15 for 1 hour and washed three times with PBS. Viral RNA and CDHR3 mRNA expression were quantified by qPCR (see below) and the presence of RV-C was examined by Western blot and immunofluorescence using anti-EV-D68 vp3. Late endosomes were visualized with anti-EEA1 (Invitrogen, Carlsbad, CA). Images were visualized using a Leica SP5 inverted laser confocal microscope (Buffalo Grove, IL).
B6.129 tlr2tm1kir j mice
Manufactured by Jackson ImmunoResearch
The B6.129-Tlr2tm1Kir/J mice are a genetically modified mouse strain that carries a targeted mutation in the Toll-like receptor 2 (Tlr2) gene. This mutation results in the disruption of the Tlr2 gene, leading to the absence of a functional Tlr2 protein. These mice are commonly used in research to study the role of Toll-like receptor 2 in various biological processes and immune responses.
Automatically generated - may contain errors
3 protocols using b6.129 tlr2tm1kir j mice
1
Macrophage Differentiation and Rhinovirus Infection
2
Inbred Mouse Models for Research
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CsA mice from our inbred colony, CB57BL/6J and B6.129 Tlr2tm1Kir/J mice were from Jackson Laboratories. The mice were maintained (≤10 in each cage) under standard conditions of temperature and light, and were fed with standard laboratory chow and water ad libitum. Adult mice were killed by cervical dislocation and newborn mice by decapitation. 298 adult and newborn mice were used for this study. The Ethical committee of Umeå University, Umeå, Sweden has approved the animal care and experiments.
3
Macrophage Differentiation and Rhinovirus Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate bone marrow-derived macrophages, mouse bone marrow monocytes were isolated from C C57BL/6J and TLR2−/− (B6.129-Tlr2tm1Kir/J) mice (Jackson Laboratories) and cultured in L929 medium, a source of macrophage colony-stimulating factor, for 7 days 49 (link). Human primary peripheral blood mononuclear cells (PBMC; Precision for Medicine, Frederick, MD) were cultured in RPMI-1640 media supplemented with 20ng/mL of recombinant human M-CSF. THP-1 human monocytic cells (ATCC) were differentiated with 5 ng/ml of phorbol 12-myristate 13-acetate as described50 . Cells were infected with RV-A1B and RV-C15 at a multiplicity of infection (MOI) of 1.0 at 33°C. To study virus entry, human THP-1-derived macrophages and mouse bone marrow-derived macrophages were incubated with RV-A1B or RV-C15 for 1 hour and washed three times with PBS. Viral RNA and CDHR3 mRNA expression were quantified by qPCR (see below) and the presence of RV-C was examined by Western blot and immunofluorescence using anti-EV-D68 vp3. Late endosomes were visualized with anti-EEA1 (Invitrogen, Carlsbad, CA). Images were visualized using a Leica SP5 inverted laser confocal microscope (Buffalo Grove, IL).
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