PBMC were isolated of fresh blood supplemented with 0.4% calcium citrate of healthy volunteers as described before 28 . 3 nM recombinant CXCL16 (Peprotech, Hamburg, Germany) or 10% patient serum diluted in RPMI was used as chemotactic stimulus within a 96‐well transwell plate (Corning, New York, NY, USA). CXCL16 neutralization was performed with the antibody AF976 from R&D Systems. Normal goat IgG (R&D Systems) served as isotype control. Random migration was analysed against non‐conditioned medium. After 3 hrs, the transwell‐insert was removed and cells fixated using 1% paraformaldehyde. Nuclei were Hoechst‐stained, and five microscopic images per well were taken. Migrated cells were automatically counted using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/ , 1997‐2014).
Recombinant cxcl16
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Recombinant CXCL16 is a chemokine protein produced using recombinant DNA technology. Its core function is to act as a chemoattractant, promoting the migration and adhesion of cells that express the CXCR6 receptor.
Automatically generated - may contain errors
4 protocols using recombinant cxcl16
1
PBMC Chemotaxis Assay with CXCL16
2
Evaluating CXCL16-induced Cell Proliferation
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LuCa cells were seeded at a density of 2 × 104/100 ul/well in 96 well plates. Next day, keeping the volume constant, cells were stimulated with different concentration (0–200 ng/ml) of recombinant CXCL16 (Peprotech). After 24 h, cell proliferation was assessed using MTT assay [79 (link)]. Briefly, MTT (20 μL of 5 mg/ml in PBS) was added in each well and plate was incubated at 37°C for 3 h. Resultant formazan crystals were dissolved in 100 μL of DMSO and absorbance was recorded in a spectrophotometer with plate reader at 570 nm. All concentrations were tested in triplicates and the experiment was repeated three times.
3
Visualizing CXCR6 Trafficking and Tumor Cell Adhesion
4
Visualizing CXCR6 Trafficking and Tumor Cell Adhesion
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To monitor the trafficking of CXCR6 after interaction with CXCL16, 5 x 103 CXCR6-GFP transduced T cells were stimulated with 10 ng/ml recombinant CXCL16 (Peprotech, Hamburg, Germany). For visualization of the receptor, in this experiment T cells were expressing CXCR6 fused to GFP via a non-cleavable 2A sequence. Receptor trafficking was imaged over a period of 1 h and membrane expression of CXCR6 was quantified by blinded validation of at least 75 representative cells per time point. Live fluorescent microscopy was conducted with a Leica SP5 AOBS confocal microscope.
To analyse the adhesion of CXCR6-transduced T cells to tumours cells, T cells were enriched by MACS sort one day before the co-culture. One day after enrichment, 5 x 103 Panc02-OVA or T110299-OVA tumour cells were co-incubated with 5 x 104 enriched CXCR6+ T cells, non-transduced T cells or a mixture of both (1:1), which were previously labelled with PKH-67 and PKH-26 (Sigma, Germany) according to the manufacturer’s instruction. To neutralize CXCL16-mediated effect, 4 μg/ml anti-CXCL16 antibody was added. Following an incubation period of 6 h, cell clusters were gently transferred to a glass-bottom dish and analysed by confocal microscopy. The amount of CXCR6+ and non-transduced T cells per cluster was quantified by blinded counting of at least 20 representative clusters for each condition.
To analyse the adhesion of CXCR6-transduced T cells to tumours cells, T cells were enriched by MACS sort one day before the co-culture. One day after enrichment, 5 x 103 Panc02-OVA or T110299-OVA tumour cells were co-incubated with 5 x 104 enriched CXCR6+ T cells, non-transduced T cells or a mixture of both (1:1), which were previously labelled with PKH-67 and PKH-26 (Sigma, Germany) according to the manufacturer’s instruction. To neutralize CXCL16-mediated effect, 4 μg/ml anti-CXCL16 antibody was added. Following an incubation period of 6 h, cell clusters were gently transferred to a glass-bottom dish and analysed by confocal microscopy. The amount of CXCR6+ and non-transduced T cells per cluster was quantified by blinded counting of at least 20 representative clusters for each condition.
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