antibody ccl21a goat polyclonal r d systems  (R&D Systems)

Journal: Frontiers in aging

Article Title: Structural and Functional Changes in Aged Skin Lymphatic Vessels.

doi: 10.3389/fragi.2022.864860

Figure Lengend Snippet: FIGURE 6 | Aging causes decreased chemokine and altered junctional protein expression by LECs. (A) Representative confocal images of ear skin sections stained for lymphatic vessels (LYVE-1) and CCL21. (B) Quantification of CCL21 immunostaining as a percentage of lymphatic vessel area. (C) qRT-PCR quantification of CCL21 mRNA expression in sorted skin LECs. (D) Representative confocal images of ear skin wholemount stained for nuclei (DAPI), VE-cadherin (upper panel, Initial lymphatics; lower, collecting lymphatics). (E) qRT-PCR quantification of VE-cadherin and (F) ZO-1 mRNA expression in sorted skin LECs. (G) Representative confocal images of ear skin sections stained for lymphatic vessels (LYVE-1) and ICAM-1 (upper panel, immunofluorescence of both; lower, ICAM-1 only). (H) qRT-PCR quantification of mRNA expression of endothelial adhesion molecules ICAM-1 and (I) p-Selectin from sorted skin LECs. n = 5 mice from each group. All quantifications are mean ± SD, unpaired Student’s t test.

Article Snippet: Antigen Antibody type Dilution Catalog number Company LYVE-1 Goat polyclonal 1:400 2125-LY R&D Systems (Minneapolis, MN) VE-cadherin Goat polyclonal 1:500 AF1002 R&D Systems LYVE-1 Rabbit polyclonal 1:200 Ab14917 Abcam (Cambridge, MA) Podoplanin Hamster monoclonal 1:500 Ab11936 Abcam α-SMA Mouse monoclonal Cy3-conjugated 1:1000 C6198 Sigma-Aldrich (Saint Louis, MO) CD31 Rat monoclonal 1:200 553370 BD Biosciences (Franklin Lakes, NJ) CD11b Rat monoclonal 1:300 557395 BD Biosciences VEGFR3/Flt-4 Goat polyclonal 1:100 AF743 R&D Systems pAkt Rabbit monoclonal 1:400 4060s R&D Systems CCL21 Goat polyclonal 1:40 AF457 R&D Systems Cleaved Caspase-3 Rabbit monoclonal 1:100 MAB 835 R&D Systems VE-Cadherin Goat polyclonal 1:500 AF1002 R&D Systems ICAM-1 Rat monoclonal 1:100 YN1/1.7.4 Ab119871 Abcam p-Selectin Rabbit monoclonal 1:500 EPR5047 Ab134047 Abcam iNOS Rabbit polyclonal 1:100 Ab3523 Abcam CD3 Rabbit polyclonal 1:200 A0452 Dako (Santa Clara, CA) Nitrotyrosine (N-Tyr) Rabbit polyclonal 1:200 BS-8551R BIOSS (Woodburn, MA) Collagen I Rabbit polyclonal 1:100 Ab34710 Abcam MacroH2A1 Rabbit polyclonal 1:100 39593 Thermo Fisher RYR1 Rabbit polyclonal 1:200 AB9078 Millipore Frontiers in Aging | www.frontiersin.org April 2022 | Volume 3 | Article 8648603 the dorsal hindlimbs of each mouse.

Techniques: Expressing, Staining, Immunostaining, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions

doi: 10.3390/ijms22084158

Figure Lengend Snippet: Design and production of the recombinant chemokines CCL19 and CCL21. ( a ) Schematic representation of the size matched expression constructs and the corresponding final fluorescently labeled chemokine proteins. A cleavable His 6 -SUMO-tag is fused to the N-terminus of mature human CCL19 and CCL21 followed by a short, flexible linker and the S6-tag for enzymatic labelling with the dye Dy 649P1 . Human CCL19-mRFP and CCL21-mRFP are included for size comparison. ( b ) Workflow overview of the chemokine production process. IMAC purification, chemokine refolding, and CIEX purification can be performed in an automated fashion on a chromatography system.

Article Snippet: Antibodies from the following sources were used for Western blotting: mouse monoclonal anti-human CCL19 (R&D Systems, Minneapolis, MN, USA, #AB361), goat polyclonal anti-human CCL21 (R&D Systems, Minneapolis, MN, USA, #AF457), polyclonal rabbit anti-goat coupled to HRP (Dako Agilent, Santa Clara, CA, USA, #P0160), polyclonal goat anti-mouse-HRP (Jackson ImmunoResearch, West Grove, PA, USA, #115-035-003).

Techniques: Recombinant, Expressing, Construct, Labeling, Comparison, Purification, Chromatography

Journal: International Journal of Molecular Sciences

Article Title: A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions

doi: 10.3390/ijms22084158

Figure Lengend Snippet: Purification and fluorescent labelling of human CCL19 and CCL21. ( a , b ) Representative Coomassie-stained SDS-PAGE gels with samples derived from the bacterial cell lysate before loading on the IMAC column, the IMAC flow through (FT), the IMAC eluate, the CIEX-C load after cleaving the His 6 -SUMO-tag, the CIEX-C flow through (FT) and CIEX-C eluate for the CCL19-S6 ( a ) and CCL21-S6 ( b ) purification steps illustrated in b. ( c , d ) Representative HPLC chromatograms displaying the volume of gradient elution of CCL19-S6 ( c ) or CCL21-S6 ( d ) from a C-18 column used to separate correctly folded from misfolded chemokines (left panels) and to separate the fluorescently labeled chemokine from unlabeled chemokine, free substrate and the Sfp-CoA-Dy 649P1 (marked with *). Protein concentration was determined by measuring its absorbance at 280 nm (OD280); fluorescence was measured at 652 nm (OD652) and illustrated in red. ( e , f ). Representative Coomassie-stained SDS-PAGE gels (upper panels) and corresponding Western blots (WB, lower panels) of the same samples of native, S6-tagged and S6-tagged, as well as fluorescently labeled CCL19 ( e ) and CCL21 ( f ).

Article Snippet: Antibodies from the following sources were used for Western blotting: mouse monoclonal anti-human CCL19 (R&D Systems, Minneapolis, MN, USA, #AB361), goat polyclonal anti-human CCL21 (R&D Systems, Minneapolis, MN, USA, #AF457), polyclonal rabbit anti-goat coupled to HRP (Dako Agilent, Santa Clara, CA, USA, #P0160), polyclonal goat anti-mouse-HRP (Jackson ImmunoResearch, West Grove, PA, USA, #115-035-003).

Techniques: Purification, Staining, SDS Page, Derivative Assay, Labeling, Protein Concentration, Fluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions

doi: 10.3390/ijms22084158

Figure Lengend Snippet: Fluorescent CCL19-S6 649P1 and CCL21-S6 649P1 efficiently elicit CCR7-mediated signaling and migration. ( a , b ) Pre-B 300-19 cells stably expressing CCR7 were stimulated with 50 nM of the indicated CCL19 ( a ) and CCL21 ( b ) chemokine variants and changes in intracellular calcium levels were recorded by flow cytometry over time. The time point of stimulation (Stim.) is indicated. Stimulation with PBS, the chemokine solvent, served as negative control. Mean values ± SD of three independent experiments are shown. ( c , d ) HeLa cells transiently transfected with CCR7-EGFP and β-arrestin2-Nluc were stimulated with 500 nM of CCL19, CCL19-S6, or CCL19-S6 649P1 ( c ), and 1.5 µM CCL21, CCL21-S6, or CCL21-S6 649P1 ( d ), respectively, and chemokine-mediated β-arrestin2 recruitment to CCR7 was determined by BRET. Mean values ± SD of three independent experiments are depicted. ( e , f ) Pre-B 300-19 cells stably expressing CCR7 were allowed to migrate towards graded concentrations of CCL19, CCL19-S6 649P1 ( e ), CCL21, or CCL21-S6 649P1 ( f ) for 3h in a Transwell migration assay. Migrated cells were counted by flow cytometry; random migration in the absence of chemokine was subtracted. Mean values ± SD of three independent experiments are shown.

Article Snippet: Antibodies from the following sources were used for Western blotting: mouse monoclonal anti-human CCL19 (R&D Systems, Minneapolis, MN, USA, #AB361), goat polyclonal anti-human CCL21 (R&D Systems, Minneapolis, MN, USA, #AF457), polyclonal rabbit anti-goat coupled to HRP (Dako Agilent, Santa Clara, CA, USA, #P0160), polyclonal goat anti-mouse-HRP (Jackson ImmunoResearch, West Grove, PA, USA, #115-035-003).

Techniques: Migration, Stable Transfection, Expressing, Flow Cytometry, Solvent, Negative Control, Transfection, Transwell Migration Assay

Journal: International Journal of Molecular Sciences

Article Title: A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions

doi: 10.3390/ijms22084158

Figure Lengend Snippet: CCL19-S6 649P1 readily and specifically binds to CCR7-expressing cells. ( a , b ) HeLa cells transiently transfected with CCR7-EGFP or its vector control (pcDNA3) were incubated at indicated temperature and time periods with 25 nM of CCL19-S6 649P1 ( a ), or CCL21-S6 649P1 ( b ). Cell associated, chemokine-derived mean fluorescence intensities (MFI) were recorded by flow cytometry. Mean values ± SD of three independent experiments are shown. ( c ) HeLa cells transiently transfected with CCR7-EGFP or its vector control (pcDNA3) were incubated at 22 °C with 25 nM of CCL19-S6 649P1 for indicated time periods. Representative dot plots of one out of three experiments depicting CCL19-S6 649P1 binding to CCR7-EGFP expressing and pcDNA3 vector control transfected cells.

Article Snippet: Antibodies from the following sources were used for Western blotting: mouse monoclonal anti-human CCL19 (R&D Systems, Minneapolis, MN, USA, #AB361), goat polyclonal anti-human CCL21 (R&D Systems, Minneapolis, MN, USA, #AF457), polyclonal rabbit anti-goat coupled to HRP (Dako Agilent, Santa Clara, CA, USA, #P0160), polyclonal goat anti-mouse-HRP (Jackson ImmunoResearch, West Grove, PA, USA, #115-035-003).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Incubation, Derivative Assay, Fluorescence, Flow Cytometry, Binding Assay

Journal: International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group

Article Title: In Situ Thermal Ablation Augments Antitumor Efficacy of Adoptive T Cell Therapy

doi: 10.1080/02656736.2019.1653500

Figure Lengend Snippet: (A) Intravascular staining of CCL21 in cLN and TdLN by iv injection of primary Ab at 6 hours after RFA or sham procedure in CT26-bearing BALB/c mice. Organs obtained 20 min later were counterstained with fluorochrome-conjugated secondary Ab to detect anti-CCL21 Ab (red) and with anti-PNAd Ab (green) to demark the position of HEV. (B) Representative histograms for experiment shown in (A). Histograms denote quantitative image analysis of the mean fluorescence intensity (MFI) for fluorescence of PNAd (from tissue section staining) and CCL21 (from intravascular staining) in PNAd+ cuboidal LN HEV. Horizontal axes, fluorescence intensity; vertical axes, pixels with each intensity; numbers in plots, MFI. (C) Intravascular staining for ICAM-1 (red) in cLN and TdLN of CT26-bearing mice at 6 hours following sham or RFA treatment. LN were counterstained with anti-PNAd Ab (green) to identify HEV. (A-C) Scale bars, 100 μm; numbers in photomicrographs and histograms indicate MFI for quantification of all pixels analyzed within the HEV for ≥ 10 fields in non-sequential cryosections for individual mice. Data are representative of 2 independent experiments.

Article Snippet: For intravascular staining, mAb specific for ICAM-1 (3E2, 50 μg/mouse, BD Biosciences); goat polyclonal Ab for CCL21 (10 μg/mouse; R&D Systems); were injected iv via the tail vein as described ( 12 , 15 , 44 , 45 ).

Techniques: Staining, IV Injection, Fluorescence

Journal: eLife

Article Title: Sphingosine 1-phosphate-regulated transcriptomes in heterogenous arterial and lymphatic endothelium of the aorta

doi: 10.7554/eLife.52690

Figure Lengend Snippet:

Article Snippet: Antibody , anti-mouse CCL21/6Ckine (goat polyclonal) , R and D systems , Cat #: AF457 RRID: AB_2072083 , 1:200 , .

Techniques: Sequencing, Purification, Agarose Gel Electrophoresis, Software

Journal: eLife

Article Title: High-endothelial cell-derived S1P regulates dendritic cell localization and vascular integrity in the lymph node

doi: 10.7554/eLife.41239

Figure Lengend Snippet: ( A ) The IHC analysis of CCL21 (white) distribution around PNAd + HEVs, and visualisation of the automated detection of PNAd + HEVs (green surface) and of the radial areas (blue) around HEVs used for the quantification of the mean fluorescent intensity of the CCL21 signal in pLNs of in Spns2 f/f (top) and Lyve1;Spns2 Δ/Δ (bottom) mice. ( B ) The mean fluorescent intensity of the CCL21 signal in distances from −5 μm to 40 μm from the outer border of HEVs (green surface in ( C )) determined in 5 μm radial areas around HEVs in pLNs of Spns2 f/f and Lyve1;Spns2 Δ/Δ mice. ( C ) qRT-PCR analysis of CCL19, CCL21 and CXCL13 expression levels in total mRNA isolated from CD45 - /CD31 + /PNAd + high-endothelial cells sorted from pLNs of Spns2 f/f and Lyve1;Spns2 Δ/Δ mice. ( D ) ELISA of the CCL21 levels of the supernatant of high-endothelial cells cultivated with or without 10 μM FTY720 (left) or 10 μM W146 (right) in vitro. Each circle represents the mean fluorescent intensity of the CCL21 signal detected around HEVs in the visual field of a micrograph ( B ), the relative chemokine expression levels in mRNA extracted from the total CD45 - /CD31 + /PNAd + high-endothelial cells ( C ), or the CCL21 protein levels detected in the supernatant of individual cell cultures ( D ) of CD45 - /CD31 + /PNAd + high-endothelial cells; bars indicate the mean. Scale bars, 50 μm ( A ). **p<0.005; ***p<0.0005 (two-tailed unpaired Student’s t -test ( B–D )). Data are representative for 18x individual sections of 2x analyzed pLNs, iLNs and bLNs per mouse pooled from three mice per group ( A, B ), three independent mRNA preparations of 2x pLNs, iLNs and bLNs per mouse pooled from five mice per group ( C ), or three independent stimulations with n = 2 to n = 4 of a total of 8x – 16x (FTY720) or 6x – 12x (W146) individual cell cultures ( D ) with total sorted high-endothelial cells from 2x pLNs, iLNs and bLNs per mouse pooled from five mice per group in vitro.

Article Snippet: Antibody , goat anti-mouse polyclonal CCL21 (AF457) , R and D Systems Inc, Minneapolis, MN , RRID: AB_2072083 , [2 μg/ml].

Techniques: Quantitative RT-PCR, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, In Vitro, Two Tailed Test

Journal: eLife

Article Title: High-endothelial cell-derived S1P regulates dendritic cell localization and vascular integrity in the lymph node

doi: 10.7554/eLife.41239

Figure Lengend Snippet:

Article Snippet: Antibody , goat anti-mouse polyclonal CCL21 (AF457) , R and D Systems Inc, Minneapolis, MN , RRID: AB_2072083 , [2 μg/ml].

Techniques: Recombinant, Control, Software

Journal: eLife

Article Title: High-endothelial cell-derived S1P regulates dendritic cell localization and vascular integrity in the lymph node

doi: 10.7554/eLife.41239

Figure Lengend Snippet:

Article Snippet: Antibody , goat anti-mouse polyclonal CCL21 (AF457) , R and D Systems Inc, Minneapolis, MN , RRID: AB_2072083 , [2 μg/ml].

Techniques: