C57bl 6 Mice, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 92/100, based on 956 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Prostaglandin E2 suppresses the differentiation of retinoic acid-producing dendritic cells in mice and humans"
Article Title: Prostaglandin E2 suppresses the differentiation of retinoic acid-producing dendritic cells in mice and humans
Journal: The Journal of Experimental Medicine
Figure Legend Snippet: Stromal-derived factors inhibit DC RALDH expression. (a and b) BM from C57BL/6 mice was cultured with GM-CSF alone or in the presence of SNs derived from primary mouse skin lines (skin), 3T3 fibroblasts, or EL-4 cells. After 3 d, LPS was added and, 18 h later, cells were stained with ALD and CD11c to measure DC RALDH activity. (a) A representative contour plot depicting the expression of CD11c and ALD is shown with inset values indicating the percentage of CD11c + DCs that are ALD + . (b) BM was differentiated, as in a, with SNs at various concentrations. Bar graphs show the mean percentages of CD11c + DCs that are ALD + with SD. Data were pooled from between 2 and 18 experiments. (c) DCs were differentiated from BM with GM-CSF alone or in the presence of skin SN. Cells were treated with LPS at day 3. 16–18 h later, DCs were purified with anti-CD11c or MHC class II microbeads and analyzed for RALDH2 expression by real-time qPCR. RALDH2 expression was normalized to GAPDH. Shown is relative expression of RALDH2 by skin SN versus control DCs, with each dot representing an individual experiment and horizontal bars the pooled mean. (d) DCs were differentiated from BM with GM-CSF alone, or in the presence of skin SN. After 3 d, LPS was added and, 18 h later, MHC class II + DCs were purified, pulsed with NP 366–374 -peptide, and used to stimulate CFSE-labeled CD8 + F5 T cells. 4 d later, the expression of CCR9 and PSL was analyzed by flow cytometry. Dot plots show the expression of homing receptors versus CFSE on live (PI − ) TCR + cells. Inset values are the mean percentage of dividing T cells positive for each receptor pooled from between 7 and 17 experiments. (e) CD8 + F5 T cells were activated as in d. After 4 d, T cells were labeled with CFSE or CTO, pooled at a 1:1 ratio, and transferred into congenic B6.Ly5.1 recipients (∼2 × 10 6 /recipient) that had been primed with oxazolone and challenged on the ear. After 16–18 h, the relative frequency of donor (CD45.2 + ) T cells primed by control versus skin SN-conditioned DCs at the various sites was determined by flow cytometry. A representative contour plot gated on live (PI − ) CD45.2 + donor T cells is shown, where control T cells are labeled with CTO and skin SN DC-primed T cells with CFSE. Inset is the mean frequency of each population pooled from three independent experiments.
Techniques Used: Derivative Assay, Expressing, Mouse Assay, Cell Culture, Staining, Activity Assay, Purification, Real-time Polymerase Chain Reaction, Labeling, Flow Cytometry, Cytometry
Figure Legend Snippet: Indomethacin administration promotes the emergence of splenic RALDH + DCs in vivo and the priming of CCR9 + T cells systemically. (a–c) 5 × 10 5 CFSE-labeled CD8 + F5 T cells were transferred into C57BL/6 mice and, 1 d later, mice received 50 µg NP 366–374 -peptide plus 10 µg LPS via i.v. tail vein injection (NPp+LPS). Indomethacin-treated mice received 100 µg indomethacin i.v. 1 h before peptide and then once daily. 60 h after NP 366–374 peptide injection, mice were sacrificed and the spleens analyzed. (a) The concentration of PGE2 was determined in spleen homogenate by ELISA. Shown is the mean with SEM from three to five mice pooled from two experiments. (b) Splenocytes were treated with ALD and stained with CD11c and CD3 (to gate out to CFSE + T cells). Shown is the mean percentage of CD3-CD11c + DCs that are ALD + in the spleen. Mean with SEM from 7–11 mice per group is shown, pooled from four experiments. (c) Splenocytes were stained with antibodies against CD8α and CCR9. Dot plots were gated on live (PI − ) CD8 + CFSE + T cells and show the expression of CCR9 and CFSE on donor F5 T cells. Inset value is the mean percentage of dividing T cells that are CCR9 + pooled from between 6 and 13 mice per group, acquired over four experiments.
Techniques Used: In Vivo, Labeling, Mouse Assay, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Expressing
Figure Legend Snippet: EP-4 deficiency enhances BM-DC RALDH expression. (a and b) BM cells from EP-KO or WT C57BL/6 and 129 mice were cultured with GM-CSF alone or in the presence of skin SN. After 3 d, LPS was added and, 18 h later, cells were stained with CD11c and ALD to measure DC RALDH activity. (a) Bar graph shows the mean percentage of CD11c + DCs that are ALD + for each mouse genotype with SEM pooled from four experiments (***, P
Techniques Used: Expressing, Mouse Assay, Cell Culture, Staining, Activity Assay
Figure Legend Snippet: Identification of factors that induce RALDH expression in vitro. (a) mLN, skin-draining pLN, or spleens from C57BL/6 mice were digested, treated with ALD, and stained with anti-CD11c antibodies for analysis by flow cytometry. A representative dot plot of ALD and CD11c expression from at least seven experiments is shown with inset values indicating the percentage of live (PI − ) CD11c + DCs that are ALD + . The mean was taken from between 11 and 23 mice. (b–d) CD11c + mLN cells sorted into ALD + and ALD − subsets. (c) The expression of RALDH2 mRNA by ALD + and ALD − DC subsets was examined by RT-PCR (values indicate molecular mass of PCR product). (d) NP 366–374 peptide–pulsed total CD11c + mLN DCs or CD11c + DCs sorted into ALD + or ALD − subsets were used to stimulate CFSE-labeled CD8 + F5 T cells in vitro. After 4 d, the expression of CCR9 on dividing T cells was analyzed by flow cytometry. The graph shows the mean percentage of dividing T cells that express CCR9 with SEM pooled from two independent experiments. (e) BM cells were cultured with FLT3-L in the presence of the indicated factors. After 3 d, LPS was added and, 18 h later, cells were treated with ALD and stained with CD11c. Shown is the percentage of CD11c + DCs that are ALD positive. The mean was taken from two experiments with SEM. (f) ALD − CD11c + DCs isolated from the mLN (mes), pLN, or spleen were cultured with GM-CSF and/or LPS for 48 h before analysis of RALDH activity by ALD staining. A representative contour plot of ALD and CD11c expression is shown. Inset values show the mean percentage of CD11c cells that are ALD + pooled from three experiments.
Techniques Used: Expressing, In Vitro, Mouse Assay, Staining, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Labeling, Cell Culture, Isolation, Activity Assay
Article Title: Exogenous Stimulation of Human Intervertebral Disc Cells in 3-Dimensional Alginate Bead Culture With BMP2 and L51P: Cytocompatibility and Effects on Cell Phenotype
Article Snippet: .. Briefly, cells were isolated by pronase (Roche, Basel, Switzerland) followed by
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Article Snippet: .. Adipose Tissue FACS Whole inguinal subcutaneous adipose depots were excised, minced briefly (5 min) with dissecting scissors, and digested for 75 mins at 37C in a
Article Title: Cardiac O-GlcNAcylation blunts autophagic signaling in the diabetic heart
Article Snippet: .. Briefly, hearts were rapidly excised from heparinized and anesthetized mice, perfused retrogradely with Ca2+ -free perfusion buffer consisting of (in mM) 0.6 KH2 PO4 , 0.6 Na2 HPO4 , 1.2 MgSO4 • 7H2 O, 0.032 phenol red, 12 NaHCO3 , 10 KHCO3 , 10 HEPES, 30 taurine, 10 2,3-butanedione monoxime, and 5.5 glucose, pH 7.46, followed by buffer containing 12.5 μM CaCl2 and 0.4 mg/ml