Journal: Nature Communications

Article Title: CD163 interacts with TWEAK to regulate tissue regeneration after ischaemic injury

doi: 10.1038/ncomms8792

Figure Lengend Snippet: ( a ) Analysis of plasma from WT and CD163 −/− mice before and after femoral ligation for TWEAK and sCD163 levels by ELISA ( n =5 per group). ( b ) Laser doppler analysis of CD163 −/− mice 3, 7, 14, 21 and 28 days after femoral ligation administered an isotype control or TWEAK blocking antibody ( n =5 per group). ( c ) Immunoblotting of CD163 −/− IL at 14 days after femoral ligation with administration of isotype control or TWEAK blocking antibody ( n =5 per group) with graphs showing quantitation of densitometry for phosphorylated/total p65 and NICD below. ( d ) Immunostaining for VE-cadherin (green) and β-dystroglycan (red) in CD163 −/− limbs with 14 day administration of TWEAK (5 or 25 μg kg −1 day −1 ) or saline (control) ( n =5 per group). Scale bars, 100 μm. The graphs show the number of VE-cadherin positive cells/myofibre and the average muscle fibre area. ( e ) Immunoblotting of CD163 −/− limbs with 14 day administration of TWEAK (5 or 25 μg kg −1 day −1 ) or saline (control) ( n =5 per group) with graphs showing quantitation of densitometry for phosphorylated p65/total p65, p52 and NICD. All bars show mean±s.e.m. * P <0.05 versus WT in a ; versus CD163 −/− control in b , c and e . P <0.05 versus other groups in d , e . Comparisons between groups were achieved using a two-sided student's t -test. For multiple group comparisons, we utilized a one-way ANOVA. If the variance ratio test ( F -test) was significant, a more detailed post hoc analysis of differences between groups was made using a Tukey–Kramer honest significance difference test.

Article Snippet: Recombinant mouse sCD163 (cat#7435 R&D Systems) and recombinant soluble Fn14/TWEAKR (cat#310-21Peprotech) were coated onto 96-well microtiter plate (Linbro MP Biomedicals) at a concentration of 1 μg ml −1 in bicarbonate/carbonate buffer (pH 9) overnight at 4 °C.

Techniques: Ligation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Western Blot, Quantitation Assay, Immunostaining, Saline

Journal: Nature Communications

Article Title: CD163 interacts with TWEAK to regulate tissue regeneration after ischaemic injury

doi: 10.1038/ncomms8792

Figure Lengend Snippet: ( a ) TWEAK (100 ng ml −1 ) was added to VEGFR1, Fn14 or CD163-coated wells. Bound protein was measured by ELISA using an antibody to detect TWEAK. ( b ) Fn14 or CD163 were coated onto 96-well plates and incubated with increasing doses of untagged TWEAK. Bound protein was measured by ELISA. ( c ) Immunoblotting of MDEC stimulated with TWEAK at 200 ng ml −1 (which activates both canonical and non-canonical NF-κB signalling) and increasing doses of recombinant mouse sCD163 for the indicated proteins. ( d ) Immunoprecipitation of serum before and after femoral ligation using anti-TWEAK antibody with immunoblotting for CD163. (The figures show a representative blot with a total of four experiments conducted.) ( e ) Immunostaining for VE-cadherin and β-dystroglycan (red) in limbs of CD163 −/− mice treated for 14 days with saline (control), TWEAK administration (25 μg kg −1 day −1 administered via Alzet pump) alone or in combination with sCD163 administration (25 μg kg −1 day −1 administered by intraperitoneal injection) ( n =5 per group). The graphs show the number of VE-cadherin positive cells per myofibre and the average muscle fibre area per group. ( f ) Immunblotting of CD163 −/− limb after 14 days of TWEAK administration (25 μg kg −1 day −1 administered via Alzet pump) alone or in combination with sCD163 administration (25 μg kg −1 day −1 administered by intraperitoneal injection) ( n =5 per group). O.D., optical density. Bar graphs show quantitation of densitometry for phosphorylated to total p65, p52 and NICD. * P <0.05 versus other groups. For multiple group comparisons, we utilized a one-way ANOVA. If the variance ratio test ( F -test) was significant, a more detailed post hoc analysis of differences between groups was made using a Tukey–Kramer honest significance difference test. For binding experiments, average of at least four well per group shown with each experiment repeated at least two times with representative results shown.

Article Snippet: Recombinant mouse sCD163 (cat#7435 R&D Systems) and recombinant soluble Fn14/TWEAKR (cat#310-21Peprotech) were coated onto 96-well microtiter plate (Linbro MP Biomedicals) at a concentration of 1 μg ml −1 in bicarbonate/carbonate buffer (pH 9) overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Recombinant, Immunoprecipitation, Ligation, Immunostaining, Saline, Injection, Quantitation Assay, Binding Assay

Journal: Nature Communications

Article Title: CD163 interacts with TWEAK to regulate tissue regeneration after ischaemic injury

doi: 10.1038/ncomms8792

Figure Lengend Snippet: After ischaemic injury, macrophages secrete TWEAK which induces tissue canonical NF-KB/Notch mediated myogenic progenitor cell proliferation. TWEAK's biological activity is tightly regulated by sCD163 binding which limits serum TWEAK levels and leads to resolution of canonical NF-κB/Notch activation at the site of injury, allowing for cell differentiation. In the absence of sCD163 (as seen in CD163-deficient mice), TWEAK levels are systemically elevated and TWEAK's biological effect is unopposed leading to prolonged activation of NF-κB and Notch signalling which causes greater myogenic progenitor cell proliferation-induced myogenesis not limited to the site of injury.

Article Snippet: Recombinant mouse sCD163 (cat#7435 R&D Systems) and recombinant soluble Fn14/TWEAKR (cat#310-21Peprotech) were coated onto 96-well microtiter plate (Linbro MP Biomedicals) at a concentration of 1 μg ml −1 in bicarbonate/carbonate buffer (pH 9) overnight at 4 °C.

Techniques: Activity Assay, Binding Assay, Activation Assay, Cell Differentiation

Journal: Inflammation

Article Title: Caveolin-2 Knockdown Alleviated Sepsis-induced Acute Lung Injury Via Promoting Macrophage M2 Polarization and Inhibiting Apoptosis by Hippo Signaling Pathway

doi: 10.1007/s10753-025-02378-2

Figure Lengend Snippet: Cav2 knockdown inhibited pulmonary apoptosis and promoted M2 polarization of sepsis-induced ALI in vivo. ( A ) Western blotting analysis of the expression of Bax, Bak, Caspase-3, Bcl-2, CD86, iNOS, CD80, CD206, CD163 and Arg1 protein in lung tissues, GAPDH serves as an internal control ( N = 3 per group). ( B , C , D , E ) The mRNA expression of Bax, Bak, Caspase-3 and Bcl-2 protein in lung tissues, detected by RT-qPCR ( N = 8 per group). ( F, G , H , I , J , K ) RT-qPCR analysis of the expression of CD86, iNOS, CD80, CD206, Arg1 and CD163 in lung tissues ( N = 8 per group). Mice were euthanized 24 h post-CLP, above data of animal models were expressed as median and interquartile range. * P < 0.05, ** P < 0.01, *** P < 0.001vs. Control group. # P < 0.05, ## P < 0.01 vs. CLP group

Article Snippet: Antibodies against Bax (14796S, CST, USA, dilution ratio of WB,1:1000, IHC,1:200), Bak (12150S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), Caspase-3 (9662S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:1000), Bcl-2 (15071S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:500), Lats1 (ab243656, Abcam, USA, dilution ratio of WB,1:1000), Mst1 (ab317410, Abcam, UK,dilution ratio of WB,1:1000), Sav1 (ab307698, Abcam, UK,dilution ratio of WB,1:1000), p -YAP (ab313464, Abcam, USA, dilution ratio of WB,1:500), YAP (HY- P86386 , MCE, USA, dilution ratio of WB,1:1000, IF,1:200), p -TAZ (Sc-17610-R, Santa Cruz, USA, dilution ratio of WB,1:200), TAZ (83669S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), iNOS (22226-1-AP, Proteintech, China, dilution ratio of WB,1:1000), CD86 (13395-1-AP, Proteintech, China,dilution ratio of WB,1:2000), CD80 (66406-1-lg, Proteintech, China, dilution ratio of WB,1:1000), CD163 (HY- P81177 , MCE, USA, dilution ratio of WB,1:2000), CD206 (32647-1-AP, Proteintech, China, dilution ratio of WB,1:2000), Arg1 (16001-1-AP, Proteintech, China, dilution ratio of WB,1:5000), GAPDH (2118S, CST, USA, dilution ratio of WB,1:1000), Histone H3 (HY- P86672 , MCE, USA, dilution ratio of WB,1:10000), VT02956 (MCE, USA), MY-875 (MCE, USA), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8001, MCE, USA, dilution ratio of WB,1:8000), HRP-conjugated AffiniPure Goat Anti-Mouse IgG H&L (HY-P8004, MCE, USA,dilution ratio of WB,1:10000), APC-anti CD80 antibody (17-0801-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), APC-anti CD206 (17-2061-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), PE-MHC II (12-5322-81, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:200), FITC-anti-CD163 (11-1631-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:50).

Techniques: Knockdown, In Vivo, Western Blot, Expressing, Control, Quantitative RT-PCR

Journal: Inflammation

Article Title: Caveolin-2 Knockdown Alleviated Sepsis-induced Acute Lung Injury Via Promoting Macrophage M2 Polarization and Inhibiting Apoptosis by Hippo Signaling Pathway

doi: 10.1007/s10753-025-02378-2

Figure Lengend Snippet: Cav2 knockdown mitigated macrophage apoptosis and promoted M2 polarization in MHS cells induced by LPS. ( A ) Western blotting analysis the expression of Bak, Bax, Caspase-3, Bcl-2, iNOS, CD86, CD80, CD206, Arg1 and CD163 proteins in MHS cells, GAPDH serves as an internal control ( N = 3 per group). ( B , C , D , E ) RT-qPCR analysis the mRNA expression of Bak, Bax, Caspase-3 and Bcl-2 protein in MHS cells ( N = 8 per group). ( F , G , H , I , J , K ) RT-qPCR analysis of the mRNA expression of CD86, iNOS, CD80, CD206, Arg1 and CD163 in MHS cells ( N = 8 per group). ( L , M ) Flow cytometry was used to detect M1 and M2 polarization related phenotype in each MHS cells groups ( N = 3 per group). Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. Control group. # P < 0.05, ## P < 0.01, ### P < 0.001vs. LPS group

Article Snippet: Antibodies against Bax (14796S, CST, USA, dilution ratio of WB,1:1000, IHC,1:200), Bak (12150S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), Caspase-3 (9662S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:1000), Bcl-2 (15071S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:500), Lats1 (ab243656, Abcam, USA, dilution ratio of WB,1:1000), Mst1 (ab317410, Abcam, UK,dilution ratio of WB,1:1000), Sav1 (ab307698, Abcam, UK,dilution ratio of WB,1:1000), p -YAP (ab313464, Abcam, USA, dilution ratio of WB,1:500), YAP (HY- P86386 , MCE, USA, dilution ratio of WB,1:1000, IF,1:200), p -TAZ (Sc-17610-R, Santa Cruz, USA, dilution ratio of WB,1:200), TAZ (83669S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), iNOS (22226-1-AP, Proteintech, China, dilution ratio of WB,1:1000), CD86 (13395-1-AP, Proteintech, China,dilution ratio of WB,1:2000), CD80 (66406-1-lg, Proteintech, China, dilution ratio of WB,1:1000), CD163 (HY- P81177 , MCE, USA, dilution ratio of WB,1:2000), CD206 (32647-1-AP, Proteintech, China, dilution ratio of WB,1:2000), Arg1 (16001-1-AP, Proteintech, China, dilution ratio of WB,1:5000), GAPDH (2118S, CST, USA, dilution ratio of WB,1:1000), Histone H3 (HY- P86672 , MCE, USA, dilution ratio of WB,1:10000), VT02956 (MCE, USA), MY-875 (MCE, USA), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8001, MCE, USA, dilution ratio of WB,1:8000), HRP-conjugated AffiniPure Goat Anti-Mouse IgG H&L (HY-P8004, MCE, USA,dilution ratio of WB,1:10000), APC-anti CD80 antibody (17-0801-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), APC-anti CD206 (17-2061-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), PE-MHC II (12-5322-81, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:200), FITC-anti-CD163 (11-1631-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:50).

Techniques: Knockdown, Western Blot, Expressing, Control, Quantitative RT-PCR, Flow Cytometry

Journal: Inflammation

Article Title: Caveolin-2 Knockdown Alleviated Sepsis-induced Acute Lung Injury Via Promoting Macrophage M2 Polarization and Inhibiting Apoptosis by Hippo Signaling Pathway

doi: 10.1007/s10753-025-02378-2

Figure Lengend Snippet: Cav2 knockdown promoted M2 polarization and attenuated macrophage apoptosis by regulating Hippo signaling pathway in vitro. ( A ) Immunofluorescence analysis assessed the nuclear expression and localization of Nucleus YAP and Nucleus TAZ. ( B ) Western blotting analysis the expression of Nucleus YAP, Nucleus TAZ, Lats1, Mst1, Sav1, Cytoplasm p-YAP, Cytoplasm p-TAZ, Bak, Bax, Caspase-3, Bcl-2, iNOS, CD86, CD80, CD206, Arg1 and CD163 proteins in MHS cells, Histone H3 and GAPDH serves as an internal control of nuclear and cytoplasmic proteins ( N = 3 per group). ( B , C ) The expression of M1 and M2 polarization related phenotype in MHS cells, detected by flow cytometry. ( D , E , F , G ) The mRNA expression of Bak, Bax, Caspase-3 and Bcl-2. ( H , I , J , K ) The mRNA expression of iNOS, CD86, CD206 and Arg1. ( L , M , N ) The mRNA expression of IL-1β, IL-6 and TNF-α in MHS cells, detected by RT-qPCR. Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. Control group. # P < 0.05, ## P < 0.01, ### P < 0.001 vs. LPS group. $ P < 0.05 vs. LPS + LV-si-Cav2 group. Ɛ P < 0.05, ƐƐ P < 0.01 vs. LPS + LV-OE-Cav2 group

Article Snippet: Antibodies against Bax (14796S, CST, USA, dilution ratio of WB,1:1000, IHC,1:200), Bak (12150S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), Caspase-3 (9662S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:1000), Bcl-2 (15071S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:500), Lats1 (ab243656, Abcam, USA, dilution ratio of WB,1:1000), Mst1 (ab317410, Abcam, UK,dilution ratio of WB,1:1000), Sav1 (ab307698, Abcam, UK,dilution ratio of WB,1:1000), p -YAP (ab313464, Abcam, USA, dilution ratio of WB,1:500), YAP (HY- P86386 , MCE, USA, dilution ratio of WB,1:1000, IF,1:200), p -TAZ (Sc-17610-R, Santa Cruz, USA, dilution ratio of WB,1:200), TAZ (83669S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), iNOS (22226-1-AP, Proteintech, China, dilution ratio of WB,1:1000), CD86 (13395-1-AP, Proteintech, China,dilution ratio of WB,1:2000), CD80 (66406-1-lg, Proteintech, China, dilution ratio of WB,1:1000), CD163 (HY- P81177 , MCE, USA, dilution ratio of WB,1:2000), CD206 (32647-1-AP, Proteintech, China, dilution ratio of WB,1:2000), Arg1 (16001-1-AP, Proteintech, China, dilution ratio of WB,1:5000), GAPDH (2118S, CST, USA, dilution ratio of WB,1:1000), Histone H3 (HY- P86672 , MCE, USA, dilution ratio of WB,1:10000), VT02956 (MCE, USA), MY-875 (MCE, USA), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8001, MCE, USA, dilution ratio of WB,1:8000), HRP-conjugated AffiniPure Goat Anti-Mouse IgG H&L (HY-P8004, MCE, USA,dilution ratio of WB,1:10000), APC-anti CD80 antibody (17-0801-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), APC-anti CD206 (17-2061-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), PE-MHC II (12-5322-81, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:200), FITC-anti-CD163 (11-1631-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:50).

Techniques: Knockdown, In Vitro, Immunofluorescence, Expressing, Western Blot, Control, Flow Cytometry, Quantitative RT-PCR

Journal: Inflammation

Article Title: Caveolin-2 Knockdown Alleviated Sepsis-induced Acute Lung Injury Via Promoting Macrophage M2 Polarization and Inhibiting Apoptosis by Hippo Signaling Pathway

doi: 10.1007/s10753-025-02378-2

Figure Lengend Snippet: Cav2 knockdown promoted M2 polarization and attenuated macrophage apoptosis by regulating Hippo signaling pathway in vivo. ( A ) Western blotting analysis the expression of Nucleus TAZ, Nucleus YAP, Lats1, Mst1, Sav1, Cytoplasm p-YAP Cytoplasm p-TAZ, Bak, Bax, Caspase-3, Bcl-2, iNOS, CD86, CD80, CD206,Arg1 and CD163 proteins in lung tissues, Histone H3 and GAPDH serves as an internal control of nuclear and cytoplasmic proteins ( N = 3 per group). ( B ) IHC results showing the expression levels of Bak, Bax, Caspase-3 and Bcl-2 (200X, Scale bar = 50 μm). ( C , D ) Percentage of IHC positive cells and positive cells counts score of IHC ( N = 8 per group). ( E ) HE staining of lung tissues (100X, Scale bar = 100 μm). ( F ) Lung tissues of HE staining injury scoring ( N = 8 per group). ( G , H , I ) The mRNA expression of IL-1β, IL-6 and TNF-α in lung tissues, detected by RT-qPCR ( N = 8 per group). Above data of animal models were expressed as median and interquartile range, * P < 0.05, ** P < 0.01, *** P < 0.001vs.Control group. # P < 0.05, ## P < 0.0 vs.CLP group. $ P < 0.05, $$ P < 0.01 vs. CLP + AAV-si-Cav2 group

Article Snippet: Antibodies against Bax (14796S, CST, USA, dilution ratio of WB,1:1000, IHC,1:200), Bak (12150S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), Caspase-3 (9662S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:1000), Bcl-2 (15071S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:500), Lats1 (ab243656, Abcam, USA, dilution ratio of WB,1:1000), Mst1 (ab317410, Abcam, UK,dilution ratio of WB,1:1000), Sav1 (ab307698, Abcam, UK,dilution ratio of WB,1:1000), p -YAP (ab313464, Abcam, USA, dilution ratio of WB,1:500), YAP (HY- P86386 , MCE, USA, dilution ratio of WB,1:1000, IF,1:200), p -TAZ (Sc-17610-R, Santa Cruz, USA, dilution ratio of WB,1:200), TAZ (83669S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), iNOS (22226-1-AP, Proteintech, China, dilution ratio of WB,1:1000), CD86 (13395-1-AP, Proteintech, China,dilution ratio of WB,1:2000), CD80 (66406-1-lg, Proteintech, China, dilution ratio of WB,1:1000), CD163 (HY- P81177 , MCE, USA, dilution ratio of WB,1:2000), CD206 (32647-1-AP, Proteintech, China, dilution ratio of WB,1:2000), Arg1 (16001-1-AP, Proteintech, China, dilution ratio of WB,1:5000), GAPDH (2118S, CST, USA, dilution ratio of WB,1:1000), Histone H3 (HY- P86672 , MCE, USA, dilution ratio of WB,1:10000), VT02956 (MCE, USA), MY-875 (MCE, USA), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8001, MCE, USA, dilution ratio of WB,1:8000), HRP-conjugated AffiniPure Goat Anti-Mouse IgG H&L (HY-P8004, MCE, USA,dilution ratio of WB,1:10000), APC-anti CD80 antibody (17-0801-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), APC-anti CD206 (17-2061-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), PE-MHC II (12-5322-81, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:200), FITC-anti-CD163 (11-1631-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:50).

Techniques: Knockdown, In Vivo, Western Blot, Expressing, Control, Staining, Quantitative RT-PCR

Journal: Acta biomaterialia

Article Title: A subtype specific probe for targeted magnetic resonance imaging of M2 tumor-associated macrophages in brain tumors.

doi: 10.1016/j.actbio.2025.01.003

Figure Lengend Snippet: Fig. 6. Immunohistochemical and FACS validations of M2pep-uIONP targeting M2 TAMs. Confocal microscopic images of tumor tissues collected from mice at 24 h after i.v. injection of probes show (A) Cy7-M2pep-uIONP were highly co-localizing with CD68- and CD163-double positive M2 TAM, revealing the targeting specificity. In comparison, the non-targeted control probe Cy7-scM2pep-uIONP and Cy7-Ferumoxytol not only exhibited much less tumoral accumulation, but also were not co-localized with M2 TAM. Scale bars: 37 µm. (B) Flow cytometric zebra plots and (C) the corresponding quantification of CD68- and CD163-double positive M2 TAM targeted by FITC-M2pep-uIONP, FITC-scM2pep-uIONP, and FITC-Ferumoxytol. The gating strategy involves sorting CD163+ M2 TAM population in live cells dissociated from mouse cerebrums by gating on CD68+ pan-macrophage populations, followed by further gating to assess the FITC-labeled nanoparticles.

Article Snippet: Rabbit polyclonal anti-CD163 antibody was purchased from Boster Biological Technology (Pleasanton, CA, USA).

Techniques: Immunohistochemical staining, Injection, Comparison, Control, Labeling

Journal: Experimental and Therapeutic Medicine

Article Title: Role of SARS‑CoV‑2 nucleocapsid protein in affecting immune cells and insights on its molecular mechanisms

doi: 10.3892/etm.2023.12203

Figure Lengend Snippet: Quantification of TNF-α, IL-10, IL-6 and CD163. The concentrations of (A) TNF-α, (B) IL-10, (C) IL-6 and (D) CD163 were determined in the supernatant of cultured Raw264.7 cells treated with the severe acute respiratory syndrome coronavirus 2 N protein. Data are presented as mean ± SD (n=3). * P<0.05 and ** P<0.01 vs. blank control.

Article Snippet: PageRuler TM Prestained protein ladder (cat. no. 26616) was purchased from Thermo Fisher Scientific, Inc. Trypsin-EDTA (0.25%) used to digest Raw264.7 cells for cell passage was purchased from Gibco; Thermo Fisher Scientific Inc. TNF-α Elisa kit (Cat. no. EK0527), IL-6 Elisa Kit (Cat. no. EK0411), IL-10 Elisa Kit (Cat. no. EK0417) and CD163 ELISA kit (Cat. no. EK1146) were purchased from Boster Biological Technology co., Ltd.).

Techniques: Cell Culture, Control

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Confocal immunofluorescence microscopy images of BALF cells from an asthmatic subject showing CD163 expression by CD68 + AMΦs. The scale bar indicates 5 μm. (B) Gating strategy for identification of human CD163 + alveolar macrophages in BALF. Cellular debris was excluded using a forward light scatter/side scatter plot and doublets were excluded using width parameter on FSC and SSC properties. CD45 + cells, that co-expressed CD14 and CD68, were identified as alveolar macrophages using side scatter and CD45 bivariate plots from which lymphocytes had been excluded. A microscopic image of sorted CD45 + /CD14 + /CD68 + /CD163 + cells shows a cellular population possessing typical cellular characteristics of alveolar macrophages. (C) MFI of cell surface CD163 expression by CD45 + /CD14 + /CD68 + AMΦs in BALF from normal individuals and asthmatic subjects (n = 7, P < 0.008, paired t test). (D) A representative histogram overlay comparing cell surface CD163 expression by AMΦs from a normal individual and an asthmatic subject.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Immunofluorescence, Microscopy, Expressing

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Intron 15–16 of the Cd163 gene was not detected by PCR of genomic DNA from Cd163 −/− mice, whereas the E. coli lacZ coding sequence from the targeting vector could only be detected in Cd163 −/− mice. Results from 2 mice are shown. (B) Cd163 mRNA was only detected by qRT-PCR in the lungs of wild type (WT), but not Cd163 −/− mice (n = 10 mice). (C) WT and Cd163 −/− mice were sensitized and challenged by daily nasal administration of HDM (25 μg) or saline, 5 days a week, for 5 weeks. End-point analysis was performed 24 h later. (D and E) Total BALF inflammatory cells (Panel D) and inflammatory cell types (Panel E) (n = 9 – 10 mice, *P < 0.05). (F) Representative lung histology from WT and Cd163 −/− mice that had been sensitized and challenged with saline or HDM were stained with hematoxylin and eosin (H & E) (x 200) or periodic acid Schiff (PAS) (x200 and x1000). (G) Serum HDM-specific IgE (n = 15 mice). (H) Quantification of MCM (n = 10 mice, *P < 0.01). ( I ) Western blots of lung proteins were reacted with antibodies against Muc5AC or β-actin. A representative blot from 6 replicate experiments is shown. (J) Relative Muc5AC expression as compared to β-actin was quantified by densitometry (n = 6, * P < 0.001). (K) Chi3L3/4 protein in BALF (n = 10 mice, *P < 0.05). Panels D, E, H and K are representative of two or three independent experiments. Panel G is pooled data from two independent experiments. Numbers of mice that were included in each experimental condition are shown.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Sequencing, Plasmid Preparation, Quantitative RT-PCR, Saline, Staining, Western Blot, Expressing

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) WT and Cd163 −/− mice were sensitized by intraperitoneal (i.p.) administration of HDM (50 μg/ml) emulsified in 2% aluminum hydroxide (ALUM) on days 1 and 4, challenged with intranasal administration of HDM (100 μg) on day 8, and end-points were analyzed on day 10. (B) BALF eosinophils (n = 10 mice, *P < 0.0001, Mann Whitney test). (C) The percentage of CD45 + /CD11c + /Siglec-F + /CD64 + /CD206 + BALF AAMs were quantified by flow cytometry (n = 10 mice, P = NS, Mann Whitney test). (D) The MFI of CD206 expression by CD45 + /CD11c + /Siglec-F + /CD64 + /CD206 + BALF AAMs was quantified by flow cytometry (n = 10 mice, P = 0.0029, Mann Whitney test). The gating strategy used to identify CD45 + /CD11c + /Siglec-F + /CD64 + BALF alveolar macrophages is presented in . Data are pooled from two independent experiments.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: MANN-WHITNEY, Flow Cytometry, Expressing

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: WT and Cd163 −/− mice were sensitized and challenged by daily nasal administration of HDM (25 μg) or saline, 5 days a week, for 5 weeks and end-points were analyzed 24 h later. (A) Cytokines in lung homogenates from HDM-challenged WT and Cd163 −/− mice (n = 18 mice). (B) Ex vivo cultures of mediastinal lymph node cells from HDM-challenged WT and Cd163 −/− mice were re-stimulated with HDM (100 μg/ml) or saline (as a control) and cytokine secretion was quantified (n = 3 – 10 mice). (C) Chemokines in BALF and lung homogenates (n = 16 – 18 mice, * P < 0.01). Data in Panels A and C are pooled from two independent experiments, while Panel B is representative of two independent experiments that showed similar results. Numbers of mice that were included in each experimental condition are shown.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Saline, Ex Vivo, Control

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Wild type (WT) and Cd163 −/− mice were sensitized and challenged by daily intranasal administration of HDM (25 μg) or saline, 5 days a week, for 5 weeks. Mice received concurrent nasal administration of a neutralizing anti-CCL24 antibody or control immunoglobulin, 3 days a week for 5 weeks. End-point analysis was performed 24 h later. ( B ) BALF eosinophils (n = 9 – 10 mice, *P < 0.0001, HDM + control antibody vs. HDM + anti-CCL24 neutralizing antibody). ( C ) Quantification of MCM (n = 6 – 10 mice, *P < 0.0001, HDM + control antibody vs. HDM + anti-CCL24 neutralizing antibody). ( D ) Representative histologic lung sections stained with hematoxylin and eosin (x 200). (E) AMΦs were isolated from HDM-challenged WT and Cd163 −/− mice, cultured ex vivo with or without HDM (500 μg/ml) and CCL24 secretion was quantified (n = 3 – 5 mice, *P < 0.01, Cd163 −/− + HDM vs. WT + HDM). Panels B & C represent pooled data from two independent experiments, while Panel E is representative data from one of two independent experiments. Numbers of mice that were included in each experimental condition are shown.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Saline, Control, Staining, Isolation, Cell Culture, Ex Vivo

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Bone marrow-derived dendritic cells (BMDCs) from WT and Cd163 −/− mice that had been pulsed with HDM or saline, as a control, were adoptively transferred to WT recipient mice on Day 10. All mice received nasal HDM challenges on days 23 through 26 and end-points were analyzed on day 27. ( B and C ) Total BALF inflammatory cells (Panel B) and inflammatory cell types (Panel C) (n = 9 – 16 mice). (D) CCL24 in BALF (n = 15 mice, P = NS, Mann-Whitney test). (E) CD11c + DCs isolated from MLNs of WT and Cd163 −/− mice that had been pulsed in vivo by intranasal administration of HDM (100 μg) or saline, as a control, were adoptively transferred to WT recipient mice. All mice received nasal HDM challenges on days 9 through 14 and end-points were analyzed on day 15. ( F and G ) Total BALF inflammatory cells (Panel F) and inflammatory cell types (Panel G) (n = 7 – 10 mice). ( H ) CCL24 in BALF (n = 10 mice, P = NS, Mann-Whitney test). Panels B – D and F – H represent pooled data from two independent experiments. Numbers of mice that were included in each experimental condition are shown. The same numbers of mice are shown for Panels B and C and Panels F and G.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Derivative Assay, Saline, Control, MANN-WHITNEY, Isolation, In Vivo

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Effector splenic CD4 + T cells were isolated from wild type (WT) mice that had been sensitized by intraperitoneal (i.p.) administration of saline or HDM (100 μg/ml) emulsified in 2% aluminum hydroxide (ALUM) on days 1 and 7. On day 14, 2 × 10 6 CD4 + T cells were purified from the spleens of sensitized mice and adoptively transferred via intraperitoneal administration to recipient WT and Cd163 −/− mice. All recipient mice received intranasal HDM challenges (50 μg) on days 19, 21, 23 and 25 and endpoints were analyzed on day 27. (B) BALF eosinophils (* P < 0.001, n = 10, recipient WT vs. recipient Cd163 −/− mice). (C) Quantification of MCM. (* P < 0.001, n = 10). Data are pooled from two independent experiments. Numbers of mice that were included in each experimental condition are shown.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Isolation, Saline, Purification

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: ( A ) Proteins in D. pteronyssinus extracts that bound to immobilized recombinant human CD163 (rhCD163) were eluted, resolved by SDS-PAGE and visualized by silver staining (Lane A). Band #6 was identified as D. pteronyssinus peptidase 1 (Der p1). Also shown are proteins that eluted from the rhCD163 resin in the absence of D. pteronyssinus extracts (Lane B), proteins in D. pteronyssinus extracts that did not bind to immobilized rhCD163 (Lane C) and rhCD163 alone (Lane D). ( B ) Immobilized rhCD163 was incubated with purified Der p1 and eluted proteins were resolved by SDS-PAGE and visualized by silver staining. A protein corresponding to the molecular weight of Der p1 was eluted from immobilized rhCD163 (Lane A). Proteins that were eluted in the absence of Der p1 (Lane B), purified Der p1 proteins that did not bind to the immobilized rhCD163 resin (Lane C) and Der p1 protein alone (Lane D) are shown. ( C ) Immobilized Der p1 was incubated with rhCD163 and eluted proteins resolved by SDS-PAGE and visualized by silver staining. A protein corresponding to the molecular weight of rhCD163 was eluted from immobilized rhCD163 (Lane A). Proteins that eluted from the immobilized rhCD163 resin in the absence of rhCD163 (Lane B), rhCD163 that did not bind to immobilized Der p1 (Lane C) and rhCD163 alone (Lane D) are shown. (D) Increasing amounts of purified Der p1 were bound to plastic and incubated with 120 ng of rhCD163 with or without supplemental calcium (5 mM). The amount of rhCD163 that bound to immobilized Der p1 increased in a dose-responsive fashion (n = 3, P < 0.0001). Binding of rhCD163 to immobilized Der p1 was calcium-dependent (n = 3, * P < 0.0001, supplemental calcium vs. no supplemental calcium). (E) 250 ng of purified Der p1 was immobilized to plastic and incubated with 120 ng of rhCD163 with or without supplemental calcium (5 mM) or EGTA (5 mM). The amount of rhCD163 that bound immobilized Der p1 was quantified (n = 4, * P < 0.0001, vs. PBS alone). (F) Immobilized Der p1 was incubated with human monocyte proteins. Bound proteins were eluted and Western blots were performed using an anti-human CD163 antibody (Lane A). For comparison, human monocyte proteins were subjected to Western blotting in Lane B. (G) Immobilized Der p1 was incubated with human monocyte proteins with or without supplemental calcium (5 mM) or EGTA (5 mM). Bound proteins were eluted and Western blotting was performed using an anti-human CD163 antibody. (H) CCL24 secretion by bone marrow-derived macrophages stimulated with Der p1 (20 μg/ml) (n=6, *P < 0.05 vs. medium alone). Data are representative of two or three independent experiments.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Recombinant, SDS Page, Silver Staining, Incubation, Purification, Molecular Weight, Binding Assay, Western Blot, Comparison, Derivative Assay

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) WT and Cd163 −/− mice were sensitized by intraperitoneal injection of Der p1 or saline on days 1 and 8 and challenged with intratracheal Der p1 on day 15. End-points were analyzed 72 h later. (B). Total BALF inflammatory cells and inflammatory cell types (n = 7 – 17 mice, *P < 0.05). (C) Representative lung histologic sections from WT and Cd163 −/− mice that had been challenged with saline or Der p1 and stained with hematoxylin and eosin (H & E) (x 200) or periodic acid Schiff (PAS) (x200 and x1000). ( D ) Quantification of MCM (n = 7 – 17 mice, *P < 0.0001). 30.6 ± 0.9 airways were counted per mouse. (E) BALF CCL24 levels (n = 5 – 17 mice, * P < 0.05). ( F ) Ex vivo cultures of mediastinal lymph node cells from Der p1-challenged WT and Cd163 −/− mice were re-stimulated with Der p1 (10 μg/ml) or saline, as a control, and cytokine secretion was quantified (n = 8). Panels B – F are pooled data from two independent experiments. Numbers of mice that were included in each experimental condition are shown.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Injection, Saline, Staining, Ex Vivo, Control

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) WT mice were sensitized by intraperitoneal (i.p.) injection of Der p1 or saline, as a control, on days 1 and 7, followed by the intranasal adoptive transfer of 1 × 10 5 AMΦ’s from naive donor WT or Cd163 −/− mice on day 14. All mice received an intratracheal Der p1 (10 μg) challenge on day 17 and end-points were analyzed on day 20. (B) Gating strategy for sorting of naive murine alveolar macrophages in BALF. Cellular debris was excluded using a forward light scatter/side scatter plot and doublets were excluded using width parameter on FSC and SSC properties. CD45 + cells, that co-expressed CD11c, Siglec-F and CD64 were identified as AMΦs. A microscopic image of sorted CD45 + /CD11c + /Siglec-F + /CD64 + cells shows a cellular population possessing typical characteristics of AMΦs. ( C ) Total BALF inflammatory cells and inflammatory cell types (n = 4 – 8 mice, * P < 0.05, donor Cd163 −/− AMΦ’s vs. donor WT AMΦ’s). ( D ) BALF CCL24 levels from Der p1- challenged recipient mice. (n = 8 mice, P = 0.0016, donor Cd163 −/− AMΦ’s vs. donor WT AMΦ’s, unpaired t test). Panels C and D are representative data from one of three independent experiments. Numbers of mice that were included in each experimental condition are shown. The same numbers of mice are shown for both analyses in Panel C.

Article Snippet: 10 μg of recombinant human CD163 protein (R&D Systems), Dermatophagoides pteronyssinus extract or purified Der p1 (Indoor Biotechnologies, Charlottesville, VA) were covalently immobilized to an aldehyde-activated 4% beaded agarose resin (Pierce Direct IP Kit, Thermo Fisher Scientific) for 2 h and blocked with 1% chicken egg white albumin (Sigma-Aldrich, St. Louis, MO).

Techniques: Injection, Saline, Control, Adoptive Transfer Assay

Journal: The EMBO Journal

Article Title: A G1‐like state allows HIV ‐1 to bypass SAMHD 1 restriction in macrophages

doi: 10.15252/embj.201696025

Figure Lengend Snippet: A, B Stimulated or unstimulated MDM were stained for classical macrophage markers (CD68, CD14) and M1 and M2 macrophage markers (CD163, CD80, CD86 and CD40) and analysed by FACS.

Article Snippet: Antibodies used were as follows: anti‐cdc2 (Cell Signaling Technology, Beverly, MA, USA); anti‐CDK2 (H‐298, Santa Cruz Biotechnology); anti‐pCDK2(Thr160) (Bioss Inc., MA, USA); anti‐CDK4 (DCS156, Cell Signaling Technology); anti‐CDK6 (B‐10, Santa Cruz Biotechnology); anti‐SAMHD1 (ab67820, Abcam, UK), beta‐actin (ab6276, abcam, UK); mouse anti‐MCM2 (BM‐28, BD Biosciences, UK); and rabbit anti‐MCM2 (SP85) from Sigma; pSAMHD1 (a kind gift from M. Benkirane) and ProSci (Poway, CA, USA); anti‐Geminin (NCL‐L‐Geminin, Leica); anti‐mouse F4/80 and CD11b (kind gift from S. Yona, UCL); anti‐human CD68, CD14, CD163, CD80, CD86, CD40 (kind gift from M. Noursadeghi).

Techniques: Staining

Journal: Human molecular genetics

Article Title: Tweak regulates astrogliosis, microgliosis and skeletal muscle atrophy in a mouse model of amyotrophic lateral sclerosis.

doi: 10.1093/hmg/ddv094

Figure Lengend Snippet: Figure 2. Tweak induces death of primary motoneurons via CD163, endocytosis and caspase-3. (A) Motoneurons were cultured for 24 h and treated with indicated

Article Snippet: The ELISA kits used were as follows: mouse IL-6 (R&D Systems), mouse Tweak and mouse CD163 (CUSABIO Biotech Co).

Techniques: Cell Culture