Journal: bioRxiv

Article Title: The Chemokine Receptor CCR8 is Not a High-affinity Receptor for the Human Chemokine CCL18

doi: 10.1101/2024.05.30.596614

Figure Lengend Snippet: (A) Cell surface levels of CCR8 expressed stably in the 4DE4 cell line maintained in 0.1 mg/mL G418. The green line denotes detection with an anti-CCR8 mAb and the solid histogram reflects isotype control staining. (B) Comparative chemotaxis responses of the same 4DE4-CCR8 line to CCL1 (blue) and CCL18 (red) and the parental 4DE4 line (maintained without G418) to CCL18 (black). (C) Inhibition of chemotactic responses to 1 nM CCL1 and 2 μM CCL18 from 4DE4-CCR8 cells (blue) and the parental 4DE4 line (black), respectively. Striped bars denote pre-incubation of cells for 30 minutes at 37 °C with 500 nM MC148. Data represent mean chemotactic indices ± S.E.M. from 3 independent experiments. * denotes p<0.05 as examined by a paired t-test.

Article Snippet: Creating more confusion, CCL18 is available commercially in different forms: CCL18(2-69) from PeproTech and CCL18(1-69) from R&D Systems ( ).

Techniques: Stable Transfection, Staining, Chemotaxis Assay, Inhibition, Incubation

Journal: bioRxiv

Article Title: The Chemokine Receptor CCR8 is Not a High-affinity Receptor for the Human Chemokine CCL18

doi: 10.1101/2024.05.30.596614

Figure Lengend Snippet: (A, B) Chemotactic responses to CCL1 (blue) and CCL18 (red) from two independently rederived clones of the 4DE4-CCR8 cell line maintained in 0.1 mg/mL G418. (C, D) Chemotactic responses to CCL1 (blue) and CCL18 (red) from L1.2 cells stably (C) or transiently (D) expressing CCR8. Data represent mean chemotactic indices ± S.E.M. from 3 (panels A -B) or 4 (panels C-D) independent experiments.

Article Snippet: Creating more confusion, CCL18 is available commercially in different forms: CCL18(2-69) from PeproTech and CCL18(1-69) from R&D Systems ( ).

Techniques: Clone Assay, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: The Chemokine Receptor CCR8 is Not a High-affinity Receptor for the Human Chemokine CCL18

doi: 10.1101/2024.05.30.596614

Figure Lengend Snippet: (A) Representative cell surface levels of CCR8 on the 4DE4-CCR8 stable cell line which were untreated (green line) or incubated with 100nM CCL1 (magenta line) for 30 minutes at 37 °C, then assayed by flow cytometry. Isotype staining of untreated cells is shown as a comparator (filled histogram). (B) Levels of CCR8 expression on the 4DE4-CCR8 cells following incubation with CCL1 (100 nM), CCL18 (1 µM), or CCL17 (100 nM) for 30 minutes at 37 °C. Data represent mean % basal CCR8 expression levels ± S.E.M. from 6 independent experiments. **** represents p<0.0001 and NS represents no significant difference compared with untreated cells, as examined by one way ANOVA and Bonferroni’s multiple comparisons test.

Article Snippet: Creating more confusion, CCL18 is available commercially in different forms: CCL18(2-69) from PeproTech and CCL18(1-69) from R&D Systems ( ).

Techniques: Stable Transfection, Incubation, Flow Cytometry, Staining, Expressing

Journal: bioRxiv

Article Title: The Chemokine Receptor CCR8 is Not a High-affinity Receptor for the Human Chemokine CCL18

doi: 10.1101/2024.05.30.596614

Figure Lengend Snippet: (A) Representative cell surface levels of CCR3 (green line) on the 4DE4-CCR3 stable cell line. Isotype control staining is shown as a shaded histogram. (B, C) Inhibition of chemotactic responses of 4DE4-CCR3 (B) and 4DE4-CCR8 (C) transfectants to a fixed 1 nM concentration of CCL11 or CCL1, respectively, by increasing concentrations of CCL18. Data shown are mean % of migrating levels ± S.E.M. from 5 independent experiments. **** represents p<0.0001 when compared with responses to buffer. (D) Binding of increasing concentrations of AF-CCL18 to 4DE4-CCR3 (green), 4DE4-CCR8 (red) and parental 4DE4 (black) cells. Data represent mean fluorescent indices ± S.E.M. from 5 independent experiments. ** represents p<0.01 when compared with buffer treatment, as examined by one way ANOVA and Bonferroni’s multiple comparisons test.

Article Snippet: Creating more confusion, CCL18 is available commercially in different forms: CCL18(2-69) from PeproTech and CCL18(1-69) from R&D Systems ( ).

Techniques: Stable Transfection, Staining, Inhibition, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: The Chemokine Receptor CCR8 is Not a High-affinity Receptor for the Human Chemokine CCL18

doi: 10.1101/2024.05.30.596614

Figure Lengend Snippet: (A) Sequences of CCL18 variants. (B) C4 reversed-phase HPLC traces for purified CCL18 variants. (C) Mass spectrometry data for the purified CCL18 variants. Expected masses assume two disulfide bonds. Observed masses were obtained from the 5+ charged peaks in electrospray ionization spectra. (D) G protein activation data for CCL18 variants (red) and CCL1 (positive control, blue), each at 100 nM. (E) The CCL18 variants (red), each at 300 nM, fail to inhibit G protein activation stimulated by 100 nM CCL1. The data in panels D and E were obtained using Flp-In CHO cells stably expressing CCR8. The presented data are mean ± S.E.M. of at least three independent experiments, each performed in triplicate.

Article Snippet: Creating more confusion, CCL18 is available commercially in different forms: CCL18(2-69) from PeproTech and CCL18(1-69) from R&D Systems ( ).

Techniques: Purification, Mass Spectrometry, Activation Assay, Positive Control, Stable Transfection, Expressing