Project description:Murine splenocytes were isolated from the spleens of C57BL/6J mice and were pretreated in vitro for three days in the presence of Pam3CSK4 (1 μg/ml), high pure LPS from E.coli O111:B4 (100 ng/ml) and R848 (5 μg/ml). PBS-treated splenocytes served as negative control. We used Qiagen Toll-like Receptor RT2 Profiler PCR Array kit to quantitate gene expression profiling of the TLR signaling pathway.
Project description:Murine splenocytes were isolated from the spleens of C57BL/6J mice and were pretreated in vitro for three days in the presence of PBS, Pam3CSK4 (1 μg/ml), high pure LPS from E.coli O111:B4 (100 ng/ml) and R848 (5 μg/ml). BALB/c irradiated recipients, were transplanted with T-cell depleted bone marrow alone, or with the aforementioned pretreated splenocytes, and 10 days after, PBMCs from all experimental groups were collected for TLR signaling pathway analysis.We used Qiagen Toll-like Receptor RT2 Profiler PCR Array kit to quantitate gene expression profiling of the TLR signaling pathway.
Project description:Murine CD3+ T-cells were immunomagnetically purified from the spleens of C57BL/6J mice and were pretreated in vitro for three days in the presence of R848 (5 μg/ml). Gene expression profile of wild type (WT) C57BL/6J TLR7-primed T-cells, was compared to unmanipulated B6 TLR7 null CD3+ Τ-cells. We used Qiagen Toll-like Receptor RT2 Profiler PCR Array kit to quantitate gene expression profiling of the TLR signaling pathway.
Project description:Follicular CD4+ T cells were enriched from pooled splenocytes in immunized C57BL/6J mice. 1 million cells per well in a 96-well V-bottom plate were incubated with 1X PBS containing 2 ug/mL Siglec-10 Fc (or Siglec-10 R119A Fc) pre-complexed with hemin-containing Protein A-APEX2 (1:2 molar ratio) for 30 min at RT, washed with 1X PBS and incubated with 500 uM biotin-tyramide (Millipore) in 1X PBS for 30 min at RT. Cells were then washed with 1X PBS and incubated with 500 uM H2O2 for 1 min at RT to initiate protein biotinylation. Reaction was quenched by washing cells 3 times with 1X PBS supplemented with 5 mM Trolox (Millipore) and 10 mM sodium ascorbate. Cell pellets were lysed in RIPA lysis buffer with protease inhibitors and underwent automated streptavidin beads enrichment (Pierce PI88817) using KingFisher Duo Prime and on-bead trypsin digestion. 10R: Sig-10 R119A Fc; 10W: Sig-10 WT Fc.
Project description:C57Bl/6J mice were treated with 4xLPS, 1xLPS, or PBS at 7 weeks of age and BM cells were collected at 11 weeks of age. BM cells were differentiated to bone marrow-derived macrophages (BMDMs) with M-CSF, and BMDMs were harvested after a secondary stimulation with LPS or PBS for 4 hours. Total RNA was extracted and analyzed for gene expressions by bulk RNA-seq
Project description:Infectious pneumonias exact an unacceptable mortality burden worldwide. Efforts to protect populations from pneumonia have historically focused on antibiotic development and vaccine-enhanced adaptive immunity. However, we have recently reported that the lungs’ innate defenses can be therapeutically induced by inhalation of a combination of synthetic TLR ligands that synergize to protect mice against otherwise lethal pneumonia. Simultaneous treatment with ligands for TLR2/6 and TLR9 conferred robust, synergistic protection against virulent Gram-positive and Gram-negative pathogens, as well as viruses. Protection is associated with rapid pathogen killing in the lungs, and pathogen killing can be induced from lung epithelial cells in isolation. Here we explore the mechanisms underlying this dramatic phenomenon by performing microarray gene expression analysis of mouse lungs treated by aerosol with PBS (sham treatment), Pam2CSK4 (TLR 2/6 ligand), ODN2395 (TLR9 ligand), or both TLR ligands. C57BL/6J mice were placed, unrestrained, in a aerosolization chamber and inhalationally exposed to 20 minute treatment with an 8 ml volume of PBS (sham), Pam2CSK4 10 ug/ml, ODN 2395 20 ug/ml, or the combination. 4 h after treatment, the mice were deeply anesthetized, their lungs were harvested, homogenized, and total RNA was extracted. Amplified cRNA was hybridized to Illumina Sentrix MouseRef-8 v2 Beadhips, labeled with Cy3, and scanned on an Illumina iScan. At least 8 unique samples were obtained per condition.
Project description:Murine CD3+ T-cells were immunomagnetically purified from the spleens of C57BL/6J mice and were pretreated in vitro for three days in the presence of R848 (5 μg/ml). Unmanipulated T-cells served as negative control. We used Qiagen Toll-like Receptor RT2 Profiler PCR Array kit to quantitate gene expression profiling of the TLR signaling pathway.
Project description:The purpose of this project is to determine changes in proteins and signaling pathways in injured tendons of C57BL/6j and MRL/MpJ mice. The MRL.MpJ mice have been reported to have a strong repair ability compared to that of C57BL/6j mice. Identifying signaling pathways in MRL/MpJ tendons that are distinct from C57BL/6j tendons will help us understand the molecular mechanisms underlying the better healing ability of MRL/MpJ mice. The samples include normal and injured Achilles tendons 4 weeks after tenotomy surgery obtained from male and female C57BL/6j and MRL/MpJ. The Achilles injury surgery was performed at 12 weeks of age. Normal tendons were obtained age, sex and strain-matched mice without surgery. The collected normal and inured tendons were subjected to proteomics.
