To make 0.5% agar gels, 2 mL 2x RPMI (made from powder) (Sigma St. Louis, Missouri, USA) was mixed with 200 µl human serum (One Lambda, Los Angles, California, USA) and 800 µl ultrapure H2O. This was prewarmed to 37°C in a water bath. 2% agar was dissolved in ultrapure H2O by bringing it to a boil in the microwave and mixing it on high speed on a vortex mixer for 20 seconds. This process was repeated four times. One mL of the agar solution was added to the prewarmed mixture to make a 0.5% agar medium solution. Of the solution, 800 µl was added to each well of a 4 well 1.5 polymer tissue culture treated chambered coverslip (Ibidi, Martinsried, Germany) that was precoated with 20% human serum in RPMI for 30 min at 37°C. To generate a uniform concentration of CCL19 (PeproTech), CCL19 was added to a final concentration of 100 ng mL-1 before letting the agar solidify. The agar was left to set for 1 hour. Next, a three-pronged bespoke autopsy punch was used to create a line of three wells, each of a three mm diameter and 2 mm apart in the agar (Supplementary Figure 2
Ccl19
Manufactured by Thermo Fisher Scientific
Sourced in United States
CCL19 is a chemokine that plays a role in the migration and homing of dendritic cells and T cells. It is commonly used in cell culture and immunology research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ccl21, by Thermo Fisher Scientific (24 mentions)
Cxcl12, by Thermo Fisher Scientific (9 mentions)
Cxcl10, by Thermo Fisher Scientific (5 mentions)
Cxcl9, by Thermo Fisher Scientific (4 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Transwell insert, by Corning (3 mentions)
Sdf 1α, by Thermo Fisher Scientific (2 mentions)
Ccl17, by Thermo Fisher Scientific (2 mentions)
Ccl22, by Thermo Fisher Scientific (2 mentions)
Icam 1 fc, by R&D Systems (2 mentions)
Chemotx system, by Neuro Probe (2 mentions)
Transwell filter, by Corning (2 mentions)
Purecol bovine collagen 1, by Advanced BioMatrix (2 mentions)
μ slide chemotaxis chamber, by Ibidi (2 mentions)
Human serum, by Ibidi (2 mentions)
Roswell park memorial institute (rpmi), by Ibidi (2 mentions)
1.5 polymer tissue culture treated chambered coverslip, by Ibidi (2 mentions)
Human serum, by Thermo Fisher Scientific (2 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (2 mentions)
54 protocols using ccl19
1
Agar-Based Microenvironment for Chemoattractant Studies
2
Fabrication of 0.5% Agar Gels
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To make 0.5% agar gels, 2 mL 2x RPMI (made from Powder) (Sigma St. Louis, Missouri, USA) was mixed with 200 µl human serum (One Lambda, Los Angles, California, USA) and 800 µl ultrapure H2O. This was prewarmed to 37°C in a water bath. 2% agar was dissolved in ultrapure H2O by bringing it to a boil in the microwave and mixing it on high speed on a vortex mixer for 20 seconds. This process was repeated four times. One mL of the agar solution was added to the prewarmed mixture to make a 0.5% agar medium solution. Of the solution, 800 µl was added to each well of a 4 well 1.5 polymer tissue culture treated chambered coverslip (Ibidi, Martinsried, Germany) that was precoated with 20% human serum in RPMI for 30 min at 37°C. To generate a uniform concentration of CCL19 (PeproTech), CCL19 was added to a final concentration of 100 ng mL -1 before letting the agar solidify. The agar was left to set for 1 hour. Next, a three-pronged bespoke autopsy punch was used to create a line of three wells, each of a three mm diameter and 2 mm apart in the agar (Supplementary Figure 1).
3
Imaging T Cell Polarization in 3D
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Glass coverslips were overlaid with 4 μg/mL rmICAM-1/Fc (R&D, 796-IC). An 1% agarose block was formed by mixing (i) one part 2x HBSS buffer (Sigma), (ii) two parts RPMI (Invitrogen) supplemented with 20% BSA (instead of FCS) (Sigma) with 2x concentrations of all other supplements used in R10 medium (see above) and (iii) one part 4% high-molecular weight agarose (Biozym Gold Agarose, 850152) in water at 52 °C. CCL19 (20 ng/ml) (Peprotech, 250-27B) was added to soluble agarose before casting. Liquid agarose was subsequently poured into a dish, covering the coated coverslip. The agarose blocks were allowed to solidify at room temperature and were equilibrated first at 4 °C for 1 h and subsequently at 37 °C and 5% CO2 for 30 min. T cells were injected under the agarose block with a micropipette and allowed to polarize for at least 30 min at 37 °C under 5% CO2 before imaging. Epifluorescence movies were recorded using the same settings as described above. Images were taken every 30 s at 6 multi-positions with NIS Elements software (Nikon Instruments). Spinning disc microscopy was performed on an inverted spinning-disc confocal microscope (Andor) using a 100x/1.4 NA objective and a 488 nm laser line in a custom-built climate chamber (37°C under 5% CO2). Time-lapse movies were recorded every two seconds.
4
T Cell Chemotactic Migration Assay
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In vitro chemotactic migration was used to evaluate T cell lymphoid tissue‐homing capacity. Briefly, CCL19 and CCL21 (300 ng mL−1 each) (PeproTech) in 100 µL culture medium were added to the lower chamber of a 96‐well transwell plate (5 µm porosity) (Corning, CLS3388). Expanded T cells (0.1 million) as indicated were seeded into the upper chamber in an 80 µL culture medium. After 5 h, the number of cells migrating to the lower chamber was counted by trypan blue exclusion.
5
Chemokine-Induced Migration Assay for DCs
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Chemokine-induced migration of VAX-DC/MM and αDC1s was performed using 24-well transwell plates with polycarbonate inserts of 5-μm pore size (Corning Costar, Cambridge, MA, USA). A total of 600 μL of culture medium (IMDM with 10 % FBS) with CCL21 (250 ng/mL, Peprotech) or CCL19 (250 ng/mL, Peprotech) was added to the bottom of the chambers. Chemokine-free culture medium served as a control. The DCs (5 × 104 cells/100 μL) were added to the upper chamber. After incubation at 37°C for 3 h, the migrated DCs (500 μL) in the bottom chamber were collected and counted using the FACSCalibur for 60 s.
6
Chemokine-Induced Migration of Mesenchymal Stem Cells
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We assessed the influence of chemokines on cell migration of MSCs using the CytoSelectTM 96-well cell migration assay (Cell Biolabs), according to the manufacturer’s instructions44 (link)–46 (link). Feeder tray wells were filled with 150 µl of medium in the presence of seven chemokines (CCL19, CCL21, CCL25, CXCL9, CXCL10, CXCL11, and CXCL12; Peprotech; all chemokines were used at a concentration of 100 ng/ml). Migration assays were run for 8 h at 37 °C in a 5% CO2 incubator. The cells were treated with a fluorescent dye (CyQuant®) and measured at 480 nm using a SpectraMax M2 Plate Reader (Molecular Devices).
7
Chemokine-Induced Cell Migration Assay
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Migration was done in 24-well plates with transwell inserts having 8-μm pore size (Corning Incorporated). 600 μl medium containing 0.5% BSA and the chemokine (200 ng/ml SDF1α, 200 ng/ml CCL19, 200 ng/ml CCL21 or none; all PeproTech) was prepared in the lower compartment. Cell suspension (5x105 cells in 100 μl) was pipetted into the insert and cells were allowed to migrate for 5 hours. Cells in the bottom part were retrieved, counted with the C6 flow cytometer (Accuri) and calculated as the percentage of the migrated cells versus total number of cells plated into the insert (original cell suspension was counted with C6 cytometer in parallel).
Time-lapse imaging was performed on 24-well plates, which were precoated with 10 μg/ml Fibronectin (Sigma-Aldrich) for 1h at room temperature. 30,000 cells were plated per well and allowed to settle for 1 hour at 37°C before measurement. Imaging was done on Olympus IX83 microscope, with 10x objective, 180 frames every 20 seconds, using Olympus CellSense dimension software. Data were analyzed using ImageJ program and CellTracker ver. 1.1 software. At least 40 cell tracks were analyzed for each sample. Graphs were plotted in GraphPad Prism 7.
Time-lapse imaging was performed on 24-well plates, which were precoated with 10 μg/ml Fibronectin (Sigma-Aldrich) for 1h at room temperature. 30,000 cells were plated per well and allowed to settle for 1 hour at 37°C before measurement. Imaging was done on Olympus IX83 microscope, with 10x objective, 180 frames every 20 seconds, using Olympus CellSense dimension software. Data were analyzed using ImageJ program and CellTracker ver. 1.1 software. At least 40 cell tracks were analyzed for each sample. Graphs were plotted in GraphPad Prism 7.
8
Establishment of Stable Chemokine-Expressing CHO Cells
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FlpInCHO cells were purchased from Thermo Fisher Scientific. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, hygromycin B, and D-luciferin were obtained from Life Technologies (Carlsbad, CA, USA). Coelenterazine h was purchased from Prolume (Pinetop, AZ, USA). Enzymes and other materials for molecular cloning were sourced from New England Biolabs (Ipswich, MA, USA). Linear 25 kDa polyethyleneimine (PEI) was from Polysciences (Warrington, PA, USA). The human chemokines CCL17, CCL19, CCL21, CCL22, CCL27 and CCL28 (1-108) were supplied by Peprotech (Cranbury, NJ, USA) and human CCL28(4-108) was from BioLegend (San Diego, CA, USA). White 96-well CulturPlates were purchased from PerkinElmer (Boston, MA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Chemokine-Induced CD4+ T Cell Migration
10
Dendritic Cell Migration Assay
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25.000 mature DC obtained or not in presence of UC-MSC were plated on top of a 5 μm pores filter (Neuroprobe ChemoTx®system) and allowed to migrate for 2 hours at 37°C towards either medium only (RPMI medium containing 0.2% BSA) or attractant chemokines (CCL19, 300 ng/ml, CCL21, 500 ng/ml, both from PeproTech). Cells remaining in the upper compartment were gently removed, the plate was centrifuged and migrated cells were manually counted.
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