We determined the acid and pepsin soluble collagen on fibroblast supernatants using the Sircol assay (Biocolor Antrim, UK). 1-1.5 × 106 cells seeded on 75 cm2 flask were used by condition. Cells were stimulated for 48 hrs with 100 ng/ml of CX3CL1 (Biolegend San Diego, USA); non-stimulated cells were also tested. After stimulation, culture medium was collected, lyophilized and reconstituted in 70 µl deionized water. The concentration of collagen in 50 µl of each sample was evaluated by the Sircol assay according to the manufacturer's instructions; each sample was run in duplicates. Samples were read at OD550 nm and at OD600 nm in an Epoch plate spectrophotometer (Biotek, Winooski, USA). Concentration of collagen was calculated with a standard curve using the Gen 5 software (Biotek).
Cx3cl1
Manufactured by BioLegend
Sourced in United States
CX3CL1 is a chemokine that plays a role in the recruitment and adhesion of leukocytes. It is expressed on the surface of activated endothelial cells and neurons, and can act as both a chemoattractant and an adhesion molecule.
Automatically generated - may contain errors
Lab products found in correlation
Epoch plate spectrophotometer, by Agilent Technologies (1 mentions)
Sircol assay, by Biocolor (1 mentions)
8 μm pore size insert, by BD (1 mentions)
Cx3cr1, by BioLegend (1 mentions)
Cx3cl1, by R&D Systems (1 mentions)
Sfasl, by R&D Systems (1 mentions)
Matrigel, by Merck Group (1 mentions)
Fetal calf serum (fcs), by BioLegend (1 mentions)
Cxcl12, by BioLegend (1 mentions)
Collagen 4, by Merck Group (1 mentions)
Sphero accucount blank particles, by Spherotech (1 mentions)
3 protocols using cx3cl1
1
Quantifying Collagen Secretion in Fibroblasts
2
Quantifying Cell Migration in Response to CX3CL1 and sFasL
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Migration was measured using 8 μm pore size inserts (BD Biosciences) in accordance with the manufacturer’s instructions. Briefly, 24-well plates were coated with Matrigel (extracellular matrix, Sigma-Aldrich) and the inserts placed into RPMI 1640 media in the presence or absence of sFasL (400 ng/mL or indicated concentrations; R and D Systems) or CX3CL1 (400 ng/mL or indicated concentrations; R and D Systems). The cells were cultured on the inserts and incubated for 24 hr at 37°C. Cell migration was measured by counting the number of cells in the bottom of each chamber, compared with the number of migrated cells in the absence of stimulant. Anti-mouse CX3CR1 monoclonal antibodies (QA16A03; Biolegend) were added at 1 hr before stimulation to block the CX3CL1–CX3CR1 interaction.
3
Transendothelial Migration Assay for CD8+ T Cells
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Ex vivo isolated ''helped'' or ''non-helped'' H-2D b /E7 49-57 -specific CD8 + T cells were plated at equal numbers (range 7500-12500 cells) in triplicate in the upper wells of a 96 well HTS 3 mm polycarbonate transwell plate (Corning) in 50 mL Yssel's serum-free medium (Gemini Bio-Products). For transendothelial migration, sEND.1 endothelial cells were grown to confluence in the upper wells. In case of invasion assays, the upper wells were coated with collagen IV (7.5 mg/cm 2 , Sigma). Cells were seeded in the presence or absence of pan-MMP GM6001 inhibitor (10 mM, Abcam), CXCR4 inhibitor AMD3100 (5 mg/ml, Sigma) or CX3CR1 inhibitor 18a (5 mg/ml, Axon Medchem, Groningen, the Netherlands). The lower wells contained 150 of IMDM (GIBCO, Life Technologies) with either FCS, CXCL12 (100 ng/ml) or CX3CL1 (100 ng/ml) (BioLegend). After 24 or 48 h, T cells that had transmigrated into the lower wells were collected and counted by flow cytometry with the use of counting beads (Sphero AccuCount Blank Particles, Spherotech Inc., Lake Forest IL). The number of repeat experiments is indicated at the end of corresponding Figure Legend.
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