Mature DCs were obtained by treating immature DCs with LPS (100 ng/ml) for 30 min and washing three times with supplemented medium. For collagen preparation, 120 µl of DCs (stock at 2 × 106 million/ml) were carefully mixed with 205 µl of bovine type I collagen (stock 6 mg/ml) (Advanced BioMatrix, San Diego, CA, USA) and 13 µl of NaHCO3 (stock 7.5%) (Sigma-Aldrich, St. Louis, MO, USA). All solutions were previously equilibrated at 4°C. Then, the sample was loaded in a custom-made poly(dimethylsiloxane) (PDMS) “collagen chamber”. The chip was then incubated at 37°C for 30 min to allow collagen polymerization. To generate the CCL21 gradient, BM-DC medium containing 200 ng/ml of CCL21 (R&D Systems, Minneapolis, MN, USA) was added outside of the chamber. The cells were imaged by phase contrast at a frequency of 1 image every 2 min using a 10× objective. Images were processed to visualize cells by subtraction of the mean image of the whole movie at every time point, to obtain white objects on a dark background. Then cells were tracked as previously described (65 (link)).
Ccl21
Manufactured by R&D Systems
Sourced in United States, United Kingdom
CCL21 is a chemokine that is involved in the migration and homeostasis of lymphocytes. It is a member of the CC chemokine family and is produced by various cell types, including endothelial cells, dendritic cells, and lymphatic endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Ccl19, by R&D Systems (23 mentions)
Cxcl12, by R&D Systems (10 mentions)
Cxcl13, by R&D Systems (8 mentions)
Transwell plate, by Corning (6 mentions)
Sphingosine 1 phosphate (s1p), by Merck Group (5 mentions)
Ccl25, by R&D Systems (2 mentions)
Hts transwell 96 well plate, by Corning (2 mentions)
Cxcl12, by Thermo Fisher Scientific (2 mentions)
Fatty acid free bsa, by Merck Group (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Ccl20, by R&D Systems (2 mentions)
Polycarbonate membrane, by Corning (2 mentions)
Icam 1, by R&D Systems (2 mentions)
β actin, by Merck Group (2 mentions)
Cxcl10, by R&D Systems (2 mentions)
Boyden chamber, by Corning (2 mentions)
24 well transwell chamber, by Corning (2 mentions)
U0126, by Bio-Techne (2 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (2 mentions)
Transwell chamber, by Corning (2 mentions)
67 protocols using ccl21
1
Tracking Mature Dendritic Cells in Collagen
2
Dendritic Cell Migration Assay
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DC migration was assessed as described by Anderson et al. (27 (link)), with minor modifications (27 (link)). Briefly, migration was measured using a transwell system (pore size 8 μm, Corning Lifesciences), mDCs (1 × 105) were added in the upper chamber, and medium, with or without CCL21 (250 ng/ml, R&D systems) in the lower chamber. After 24 h-incubation at 37°C migrated mDCs in the lower chamber were harvested and counted using a Neubauer chamber (Celeromics). Migration efficiency was calculated from the percentage of the input cells that had migrated. The percentage of mDCs which had migrated toward the medium without CCL21 served as a negative control. The migration conditions used in this study were pre-optimized (Supplementary Figure S2 ).
3
Immunohistochemical Analysis of Intestinal Chemokine Receptors
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Small intestine tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 3.5μm slices. Followed deparaffinized, rehydrated and antigen retrieval, endogenous peroxidase activitywas blockedwith 3% H2O2. Primary antibodies of CCR7 (Abcam, USA), CCR9 (Abcam, USA), CCL21 (R&D Systems, USA), and CCL25 (R&D Systems, USA) were incubated at 4°C overnight. After washing, the slides were incubated with apporiate biotin-labeled secondary antibodies followed by staining with 3, 3’-diaminobenzidine (DAB), and counterstaining with hematoxylin. The stained slides were examined by light microscopy.
4
Histological and Cellular Analysis of Immune Cell Phenotypes
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For the histological portion of the study, paraffin-embedded sections were stained with hematoxylin and eosin. Toluidine blue or Giemsa staining was used to detect mast cells or neutrophils, respectively. The stained cells were counted in 5 different areas, and average positive numbers were calculated on mm2 per skin area.
For cell polarization and AQP9 expression, cells were cultured on glass coverslips and stimulated with fMLP (Sigma), CCL19 (R&D, MN, USA), or CCL21 (R&D, MN, USA). Cells were fixed with 4% formalin in PBS, permeabilized with 0.1% saponin, and stained with phalloidine-Alexa488 (Invitrogen), and AQP9 (Alpha diagnostic international, San Antonio, Texas, USA), anti-rabbit secondary antibody (FITC, Sigma). Frozen LN sections were stained with anti-Ly6G/Alexa594 (clone 1A8, BioLegend) and anti-CD8/FITC (clone 16-10A1, eBioscience).
For cell polarization and AQP9 expression, cells were cultured on glass coverslips and stimulated with fMLP (Sigma), CCL19 (R&D, MN, USA), or CCL21 (R&D, MN, USA). Cells were fixed with 4% formalin in PBS, permeabilized with 0.1% saponin, and stained with phalloidine-Alexa488 (Invitrogen), and AQP9 (Alpha diagnostic international, San Antonio, Texas, USA), anti-rabbit secondary antibody (FITC, Sigma). Frozen LN sections were stained with anti-Ly6G/Alexa594 (clone 1A8, BioLegend) and anti-CD8/FITC (clone 16-10A1, eBioscience).
5
Investigating Cellular Signaling Pathways
6
Chemotaxis Assay for Immune Cell Migration
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Recombinant human CXCL12 and CCL21 were purchased from R&D Systems. A transwell cell migration assay was performed using a 24‐well chemotaxis chamber with 5‐µm pore inserts precoated with 20 µg/mL fibronectin (Sigma‐Aldrich) for 1 hour at room temperature. The cells (1 × 105 cells/mL) were resuspended in DMEM/F12 supplemented with 2% FCS and added to the upper chambers, whereas CXCL12 or CCL21 to the lower chambers. After incubating for 18 hours, the cells on the lower surface of the membrane were fixed with methanol and stained with hematoxylin and eosin. Cell number was counted under a light microscope in eight fields per membrane at a 200‐fold magnification. The relative chemotactic index was determined as the ratio of the proportion of cells that migrated in response to multiple chemokines to the proportion that migrated in response to a single chemokine. The assay was performed in triplicate. Data were expressed as the mean ± SD and were analyzed by Student's t test. A two‐tailed P‐value of 0.05 or less was considered significant.
7
Flow Cytometry-based Conjugation Assay
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For flow cytometry–based conjugation assays, BMDCs were labeled with PKH26 (Sigma) and pulsed with 10 µM antigen for 2 h. T cells were labeled with Cell Proliferation Dye eFluor670 (eBioscience). T cells and BMDCs were mixed at a 1:1 ratio for the indicated times, and conjugate frequencies were determined from the percentage of T cell–APC conjugates to the total number of T cells. All conjugate assays were normalized to no antigen controls (PBS-pulsed BMDCs). For image-based conjugation studies, 10 µM peptide-pulsed BMDCs adhered to a Delta T dish coated with ICAM-1 and 2 µg CCL21 (R&D) for 1 h before imaging. T cells were added at a 1:1 ratio for the indicated times, and conjugate frequencies were determined from live imaging. All conjugation studies were normalized to no antigen controls (PBS-pulsed BMDCs).
8
In vitro Chemotaxis Assay for Immune Cells
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CCL21 and CXCL13 were purchased from R&D Systems. S1P was purchased from Sigma. In vitro migration assay was conducted using 96-well chemoTx chamber with 5-μm pore inserts (Neuroprobe) as described previously71 (link). In brief, single cells prepared from mouse spleen or lymph nodes were suspended at 8 × 106/ml in RPMI 1640 containing 1 mg/ml BSA and 20 mM HEPES (pH 7.4), and applied to upper wells (25 μl/well). The same medium without or with CCL21, CXCL13, or S1P at indicated concentrations was applied to lower wells (29 μl/well). After 1 h at 37 °C, the content of each lower well was transferred to a polypropylene pointed-bottom tube. The cells were pelleted by centrifugation at 200 × g for 5 min, resuspended in 0.1% BSA in PBS, and stained with APC-Cy7-labeled anti-mouse CD4, PerCP-Cy5.5-labeled anti-mouse CD8, and FITC-labeled anti-mouse CD19. After washing, cells were analyzed on a FACSFortessa (BD Biosciences) and analyzed with FlowJo (FlowJo, LLC). All assays were done in duplicate.
9
Transwell Migration Assay of T Cell Subsets
10
Motility Dynamics of Macrophages and Osteoclasts
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WT, Ebi2GFP/GFP, and Ch25h−/− BMDMs were cultured on 35-mm glass-bottomed dishes (MatTek) for 2 d before imaging in 30% L929-conditioned media at 37°C with 5% CO2. In vitro–differentiated OCs from the same mice were cultured on 35-mm glass-bottomed dishes for 4 d before imaging in OC differentiation media containing M-CSF and 100 ng/ml RANKL (R&D Systems) at 37°C with 5% CO2. For motility recovery experiments, WT or Ebi2GFP/GFP BMDM media were removed after 2 d and replaced with media containing titrated concentrations of CCL21 (R&D Systems), and cells were imaged 1 h after media change. Images were collected by time-lapse microscopy (Vivaview; Olympus) every 2 or 3 min for BMDMs or OCs, respectively, for 3 h total (objective 20×) at 37°C with 5% CO2. The images from four to five fields for each sample were collected and analyzed with Imaris software (Bitplane).
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