Journal: Cell Reports Methods

Article Title: Profiling migration of human monocytes in response to chemotactic and barotactic guidance cues

doi: 10.1016/j.crmeth.2024.100846

Figure Lengend Snippet: A MAP chip for quantitative analysis of immune cell migration characteristics at a single-cell level (A) The MAP chip contains four different sets of microchannels: chemotaxis, chemotactic maze, dual taxis, and distance dual taxis. Monocytes are loaded into the CENTRAL chamber of a primed MAP chip and can migrate through the microchannels into the CHEMOKINE chambers. (B) An experimental pipeline illustrating how human monocytes from different donor groups were isolated, stimulated if applicable, and loaded into the MAP chip primed with either CCL2 or CCL5 chemokine. Cell motility was then tracked at a single-cell resolution using time-lapse imaging. The data were quantified to detail monocyte migration characteristics. Additionally, flow cytometry was performed on the monocytes to quantify specific receptor expression levels.

Article Snippet: CC motif chemokine Ligand 2 (CCL2) (R&D Systems, 279-MC-010) or CCL5 (R&D Systems, 278-RN-010) was reconstituted in IMDM +20% FBS to a final concentration of 100 nM.

Techniques: Migration, Chemotaxis Assay, Isolation, Imaging, Flow Cytometry, Expressing

Journal: Cell Reports Methods

Article Title: Profiling migration of human monocytes in response to chemotactic and barotactic guidance cues

doi: 10.1016/j.crmeth.2024.100846

Figure Lengend Snippet: IFN-γ stimulation, not aging, significantly diminishes monocyte CCL2 chemotaxis (A) Schematic showing the chemotaxis microchannel design, which establishes a chemotactic gradient along a 400 μm channel for directional migration of monocytes. (B) Representative time-lapse confocal microscopy imaging of monocytes (cell membrane, green; nuclei, blue) in the chemotaxis microchannels primed with 100 nM CCL2 chemokine. Scale bar, 10 μm. (C and D) Representative imaging with tracks showing monocyte migration in the chemotaxis microchannels in the presence of CCL2 chemokine (C) or control of media (D). Scale bar, 100 μm. (E and F) Quantification of human monocyte migration (E) and migration velocity (F) in response to 100 nM CCL2 chemokine ( n = 10 deidentified healthy donors). (G) The number of migrating monocytes in the presence of CCL2 gradient or evenly distributed chemokine ( n = 6 independent MAP chips). (H and I) Quantification of human monocyte migration (H) and migration velocity (I) from 19- to 27-year-old ( n = 4–5) and 50- to 60-year-old ( n = 5 - 6) healthy donors. (J) Quantification of human monocyte migration and migration velocity upon stimulation with GM-CSF or IFN-γ ( n = 8–16 donors for control and n = 6–8 donors for stimulated monocytes). p values are from Mann-Whitney test (E and H), Welch’s t test (G and I), one-way ANOVA (J, left), and Brown-Forsythe and Welch ANOVA tests (J, right). Boxplots show the median and the range between the 25th and 75th percentiles. The whiskers stretch from the minimum and maximum values.

Article Snippet: CC motif chemokine Ligand 2 (CCL2) (R&D Systems, 279-MC-010) or CCL5 (R&D Systems, 278-RN-010) was reconstituted in IMDM +20% FBS to a final concentration of 100 nM.

Techniques: Chemotaxis Assay, Migration, Confocal Microscopy, Imaging, Membrane, Control, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: Profiling migration of human monocytes in response to chemotactic and barotactic guidance cues

doi: 10.1016/j.crmeth.2024.100846

Figure Lengend Snippet: Monocytes migrate through the path of lower hydraulic resistance regardless of donor age or proinflammatory cytokine stimulation (A) The dual-taxis microchannels contain a chemotactic gradient and varying hydraulic resistances. At the bifurcation of each microchannel, the hydraulic resistance is increased by 6×, 14×, or 51× in the right path compared to the left path. (B) Schematic showing a magnified view of the bifurcation within the 6× hydraulic resistance design. (C) Representative time-lapse confocal microscopy imaging of monocytes (cell membrane, green; nuclei, blue) in the dual-taxis microchannels primed with 100 nM CCL2 chemokine. Scale bar, 10 μm. (D) The number of migrating monocytes in the presence of CCL2 gradient or evenly distributed chemokine ( n = 6 independent MAP chips). (E–I) The migration ratio of monocytes in response to 100 nM CCL2 chemokine and varying hydraulic resistances ( n = 17–18 healthy unstimulated, n = 3–5 19- to 27-year-old, n = 6 50- to 60-year-old, n = 7 GM-CSF-stimulated donors, and n = 7–8 IFN-γ-stimulated donors). (J–L) The migration ratio of monocytes in response to 100 nM CCL5 chemokine and varying hydraulic resistances ( n = 6–8 healthy unstimulated, n = 7 GM-CSF-stimulated, and n = 7–8 IFN-γ-stimulated donors). p values are from Welch’s t test (D) and one-sample t and Wilcoxon tests (E–L). Boxplots show the median and the range between the 25th and 75th percentiles. The whiskers stretch from the minimum and maximum values.

Article Snippet: CC motif chemokine Ligand 2 (CCL2) (R&D Systems, 279-MC-010) or CCL5 (R&D Systems, 278-RN-010) was reconstituted in IMDM +20% FBS to a final concentration of 100 nM.

Techniques: Confocal Microscopy, Imaging, Membrane, Migration

Journal: Cell Reports Methods

Article Title: Profiling migration of human monocytes in response to chemotactic and barotactic guidance cues

doi: 10.1016/j.crmeth.2024.100846

Figure Lengend Snippet: Spatial confinement does not dictate the migration of human monocytes toward lower hydraulic resistance paths (A) The distance dual-taxis microchannels contain equal dimensions at the bifurcation and varying distances to a change in width and, consequentially, hydraulic pressure. The left channel has lower hydraulic resistance, and the right path has a 9.2×, 7.6×, 5.3×, or 3.5× greater hydraulic resistances. (B) Schematic showing a monocyte experiencing equal spatial confinement at the bifurcation but different hydraulic resistances. (C) Representative time-lapse confocal microscopy imaging of monocytes (cell membrane, green; nuclei, blue) in the distance dual-taxis microchannels primed with 100 nM CCL2 chemokine. Scale bar, 10 μm. (D) The number of migrating monocytes in the presence of CCL2 gradient or evenly distributed chemokine ( n = 6 independent MAP chips). (E–G) The migration ratio of monocytes in response to CCL2 and varying hydraulic resistances ( n = 17–18 healthy unstimulated, n = 4–5 19- to 27-year-old, and n = 6 50- to 60-year-old donors). (H and I) Quantification of the migration velocity of monocytes from 19- to 27-year-old ( n = 5) and 50- to 60-year-old donors ( n = 5) in response to 100 nM CCL2 chemokine. (J and K) The migration ratio of stimulated monocytes in response to CCL2 and varying hydraulic resistances ( n = 8 GM-CSF-stimulated and n = 7–8 IFN-γ-stimulated donors). (L–N) The migration ratio of stimulated monocytes in response to CCL5 and varying hydraulic resistances ( n = 8 healthy unstimulated, n = 7–8 GM-CSF-stimulated, and n = 8 IFN-γ-stimulated donors). (O and P) Quantification of monocyte migration velocity toward (O) 100 nM CCL2 or (P) CCL5 chemokine when unstimulated ( n = 16 donors for CCL2 and n = 6 donors for CCL5), GM-CSF stimulated ( n = 6 donors for CCL2 and CCL5), or IFN-γ stimulated ( n = 4 donors for CCL2 and n = 6 for CCL5). p values are from Mann-Whitney test (D and I), one sample t and Wilcoxon tests (E–G and J–N), Welch’s t test (H), and two-way ANOVA (O and P). Boxplots show the median and the range between the 25th and 75th percentiles. The whiskers stretch from the minimum and maximum values.

Article Snippet: CC motif chemokine Ligand 2 (CCL2) (R&D Systems, 279-MC-010) or CCL5 (R&D Systems, 278-RN-010) was reconstituted in IMDM +20% FBS to a final concentration of 100 nM.

Techniques: Migration, Confocal Microscopy, Imaging, Membrane, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: Profiling migration of human monocytes in response to chemotactic and barotactic guidance cues

doi: 10.1016/j.crmeth.2024.100846

Figure Lengend Snippet: IFN-γ stimulation, not aging, hinders human monocyte chemotaxis through complex pathways (A) Schematic showing the chemotactic maze with several decision-making forks. (B) Representative time-lapse confocal microscopy imaging of monocytes (cell membrane, green; nuclei, blue) in the chemotactic maze primed with 100 nM CCL2 chemokine. Scale bar, 10 μm. (C) The number of migrating monocytes in the presence of CCL2 gradient or evenly distributed chemokine ( n = 6 independent MAP chips). (D–G) Quantification of normalized monocyte migration (D), track duration (E), track length (F), and migration velocity (G) in response to 100 nM CCL2 chemokine for 19- to 27-year-old ( n = 3–5) and 50- to 60-year-old ( n = 6) donors. (H–K) Quantification of normalized monocyte migration (H), track duration (I), track length (J), and migration velocity (K) in response to 100 nM CCL2 chemokine for unstimulated ( n = 17–18 donors), GM-CSF-stimulated ( n = 8 donors), and IFN-γ-stimulated ( n = 8 donors) monocytes from deidentified donors. (L–O) Quantification of normalized monocyte migration (L), track duration (M), track length (N), and migration velocity (O) in response to 100 nM CCL5 chemokine for unstimulated ( n = 8 donors), GM-CSF-stimulated ( n = 7–8 donors), and IFN-γ-stimulated ( n = 8 donors) monocytes from deidentified donors. p values are from Mann-Whitney test (C), Welch’s t test (D–G), Brown-Forsythe and Welch ANOVA tests (H and L), Kruskal-Wallis test (I, J, M, and N), and one-way ANOVA (K and O). Boxplots show the median and the range between the 25th and 75th percentiles. The whiskers stretch from the minimum and maximum values.

Article Snippet: CC motif chemokine Ligand 2 (CCL2) (R&D Systems, 279-MC-010) or CCL5 (R&D Systems, 278-RN-010) was reconstituted in IMDM +20% FBS to a final concentration of 100 nM.

Techniques: Chemotaxis Assay, Confocal Microscopy, Imaging, Membrane, Migration, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: Profiling migration of human monocytes in response to chemotactic and barotactic guidance cues

doi: 10.1016/j.crmeth.2024.100846

Figure Lengend Snippet:

Article Snippet: CC motif chemokine Ligand 2 (CCL2) (R&D Systems, 279-MC-010) or CCL5 (R&D Systems, 278-RN-010) was reconstituted in IMDM +20% FBS to a final concentration of 100 nM.

Techniques: Blocking Assay, Recombinant, Modification, Saline, Isolation, Selection, Staining, Electron Microscopy, Software

Journal: bioRxiv

Article Title: Immune disease dialogue of chemokine-based cell communications as revealed by single-cell RNA sequencing meta-analysis

doi: 10.1101/2024.07.17.603936

Figure Lengend Snippet: a) Pan CD8 T cells migrated towards CXCL2 (at all dilutions for donor 1, and at 1 ng/ml and 10 ng/ml for donor 2), b) pan monocytes did not migrate towards RARRES2, c) pan monocytes migrated towards CCL8 (at 0.1 ng/ml, 1 ng/ml and 100 ng/ml for donor 1, and at 0.1 ng/ml and 100 ng/ml for donor 2).* p < 0.05. ** p < 0.01, *** p < 0.0001 compared to the medium control condition per donor.

Article Snippet: For the monocyte experiments, two positive controls were used; 5% (v/v) cobra venom activated human complement serum (CAS; Complement Technology Inc, cat. NC1769554), as well as CC motif chemokine ligand 8 (CCL8; R&D Systems, cat. 281-CP-010).

Techniques: Control

Journal: bioRxiv

Article Title: Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma

doi: 10.1101/2023.10.04.560856

Figure Lengend Snippet: M1 and M2 macrophages were co-cultured with BLM melanoma cells for transcriptomic (24 h) and secretomic (72 h) analyses. (A) Gene set enrichment analyses (GSEA) comparing 24 hours exposed vs un-exposed isolated cells, as indicated (n=3 donors). Normalized Enrichment Score (NES) values from the main upregulated hallmark_pathwaysfor all groups,are shown. GOMF and KEGG gene sets were additionally studied for cytokine/ chemokine inflammatory pathways. False Discovery Rates are represented (FDR q value <0.01). (B) Representative scheme highlighting the coincident most upregulated secreted chemokines (fold >4 to both unex-posed macrophages and melanoma cells). (C) Secretomic analysis (38 chemokines) of 72 hours co-cultured supernatants comparing M1+ BLM or M2+BLM with unexposed macrophages (x-axis) or BLM (y-axis) cells. Fold logarithmic average changes are shown (n=4 donors). (D) CCL8 and CCL15 secretion assessed at 72 h macrophages ±BLM conditioned media. Mean ±standard deviation (SD) values are shown (n= 5 do-nors). (E) CCL8 and CCL15 mRNA expression levels in mutually conditioned isolated cells. Mean ±SD values relative to TBP mRNA are shown (n= 4). Significant differences to the respective untreated controls, are shown (*p< 0.05, unpaired t-test).

Article Snippet: Serum-deprived media ±recombinant human CCL8 or CCL15 (R&D Systems, Minnneapolis, MN) and ±5 μg/ml neutralizing antibodies for CCR1, CCR3 and CCR5 (supplementary material, Table S1) were added on top of the gels, and spheroids were allowed to invade for 72 hours.

Techniques: Cell Culture, Isolation, Standard Deviation, Expressing

Journal: bioRxiv

Article Title: Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma

doi: 10.1101/2023.10.04.560856

Figure Lengend Snippet: (A) Serum deprivation survival assays of BLM, A375 and Skmel-103 melanoma cell lines in response to increasing concentra-tions of CCL8 and CCL15 chemokines (ng/ml), and to FCS 1%, as positive control. Mean ±SD optical density (OD) values, are shown (n= 4). (B) Proliferation response of melanoma cell lines to 200 ng/ml exogenous CCL8 and CCL15, and to FCS 1% as positive control. Mean ±SD percentages of BrdU+ nuclei, are shown (n= 3). (C) BLM spheroids embedded in 3D-collagen and allowed to invade for 72 h in the presence of increasing concentrations of exogenous CCL8 and CCL15, as indicated (ng/ml). Functional chemotactic axes were identified by using neutralizing antibodies against CCR1, CCR3, CCR5 and their corresponding control isotypes (5 μg/ml). Mean ±SD fold-invasion values, are shown (n= 3-6). Images show representative collagen-embedded spheroids showing basal and CCL15 stimulated invasion. Scale bar, 150 μm. (D) Immunoblots showing CCR1, CCR3 and CCR5 expression in whole-lysates of three melanoma cell lines and isolated CD14+ monocytes. (E) Serum deprivation survival and 3D-collagen invasion assays of monocytes in response to 100 ng/ml exogenous chemokines, including FCS 1%, as positive control (n= 5 donors). Significant differences to the respective untreated controls, are shown (*p< 0.05, paired t-test).

Article Snippet: Serum-deprived media ±recombinant human CCL8 or CCL15 (R&D Systems, Minnneapolis, MN) and ±5 μg/ml neutralizing antibodies for CCR1, CCR3 and CCR5 (supplementary material, Table S1) were added on top of the gels, and spheroids were allowed to invade for 72 hours.

Techniques: Positive Control, Functional Assay, Control, Western Blot, Expressing, Isolation

Journal: bioRxiv

Article Title: Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma

doi: 10.1101/2023.10.04.560856

Figure Lengend Snippet: TAM (A) and tumor cell (B) average MFI of CCL15, CCL8, CCR1, CCR3 and CCR5 in non-metastasizing and metastasizing primary tumors.Mann-Whitney statistical analysis was used to compare non-metastasizing vs metastasizing melanomas; p-values are shown.(C) FFPE human melanoma samples stained for CD68 (TAM marker, red) and CCL15, CCL8, CCR1, CCR3 or CCR5 (green). Scale bar, 50 μm.

Article Snippet: Serum-deprived media ±recombinant human CCL8 or CCL15 (R&D Systems, Minnneapolis, MN) and ±5 μg/ml neutralizing antibodies for CCR1, CCR3 and CCR5 (supplementary material, Table S1) were added on top of the gels, and spheroids were allowed to invade for 72 hours.

Techniques: MANN-WHITNEY, Staining, Marker

Journal: bioRxiv

Article Title: Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma

doi: 10.1101/2023.10.04.560856

Figure Lengend Snippet: (A)Disease-free and overall survival 10-year Kaplan–Meier curves. Youden’s index was used to choose a cutoff point to classify the 67 primary melanomas as ‘low’ or ‘high’: TC CCL8 (MFI, 80 a.u.), TC CCL15 (98a.u.),TAM CCL15 (69 a.u.) and TAM CCR3 (78 a.u.). Log-rank p values, are shown. (B) Summarizing model, created with BioRender.com .

Article Snippet: Serum-deprived media ±recombinant human CCL8 or CCL15 (R&D Systems, Minnneapolis, MN) and ±5 μg/ml neutralizing antibodies for CCR1, CCR3 and CCR5 (supplementary material, Table S1) were added on top of the gels, and spheroids were allowed to invade for 72 hours.

Techniques: