recombinant human ccl8  (Revvity)

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: Cancers

Article Title: Lactate-Induced CCL8 in Tumor-Associated Macrophages Accelerates the Progression of Colorectal Cancer through the CCL8/CCR5/mTORC1 Axis

doi: 10.3390/cancers15245795

Figure Lengend Snippet: Primer sequences.

Article Snippet: Recombinant human CCL8 (rCCL8) protein, macrophage colony-stimulating factor (MCSF), and Maraviroc (selective antagonist of CCR5) were purchased from MCE.

Techniques: Sequencing

Journal: Cancers

Article Title: Lactate-Induced CCL8 in Tumor-Associated Macrophages Accelerates the Progression of Colorectal Cancer through the CCL8/CCR5/mTORC1 Axis

doi: 10.3390/cancers15245795

Figure Lengend Snippet: Antibody list.

Article Snippet: Recombinant human CCL8 (rCCL8) protein, macrophage colony-stimulating factor (MCSF), and Maraviroc (selective antagonist of CCR5) were purchased from MCE.

Techniques:

Journal: Cancers

Article Title: Lactate-Induced CCL8 in Tumor-Associated Macrophages Accelerates the Progression of Colorectal Cancer through the CCL8/CCR5/mTORC1 Axis

doi: 10.3390/cancers15245795

Figure Lengend Snippet: CCL8 was induced by lactate in macrophages. ( A , B ) KEGG pathway and volcano map and volcano map analysis of differential genes on lactate-treated macrophages for 24 h. ( C ) qRT-PCR in macrophages stimulated with CM3 or LA for 24 h. ( D ) ELISA detection of CCL8 in the serum of healthy and CRC donors. ( E , F ) Representative images and quantitative analysis of immunohistochemistry for CCL8 and CD68 in CRC tissues and adjacent nontumor tissues. All t -tests were two-tailed. Mean ± SEM. ** p < 0.01; *** p < 0.001. Scale bar: 100 μm.

Article Snippet: Recombinant human CCL8 (rCCL8) protein, macrophage colony-stimulating factor (MCSF), and Maraviroc (selective antagonist of CCR5) were purchased from MCE.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Two Tailed Test

Journal: Cancers

Article Title: Lactate-Induced CCL8 in Tumor-Associated Macrophages Accelerates the Progression of Colorectal Cancer through the CCL8/CCR5/mTORC1 Axis

doi: 10.3390/cancers15245795

Figure Lengend Snippet: RNA sequencing analysis of M2-macrophage-related chemokines in macrophages treated with lactate for 24 h.

Article Snippet: Recombinant human CCL8 (rCCL8) protein, macrophage colony-stimulating factor (MCSF), and Maraviroc (selective antagonist of CCR5) were purchased from MCE.

Techniques: RNA Sequencing

Journal: Cancers

Article Title: Lactate-Induced CCL8 in Tumor-Associated Macrophages Accelerates the Progression of Colorectal Cancer through the CCL8/CCR5/mTORC1 Axis

doi: 10.3390/cancers15245795

Figure Lengend Snippet: ( A ) Western blot analysis of CCR5 in CRC tissue and adjacent nontumor tissue. ( B , C ) Representative images and quantitative analysis of the colony assay in HCT-116 and RKO cells treated with PBS, CCL8, or CCL8+Maraviroc for 15 days. ( D , E ) Representative images of the immunohistochemistry for skeleton proteins (F-actin and p-SMAD2) in RKO and HCT-116 cells treated with PBS, CCL8, or CCL8+Maraviroc for 24 h. ( F ) Western blot analysis of EMT-related proteins (PCNA, vimentin, N-cadherin) in RKO cells stimulated with CCL8, Maraviroc, or both for 24 h. ( G – K ) Representative images and quantitative analysis of subcutaneous and lung metastasis models of Balb/c-nude tumor-bearing mice, treated with PBS, CCL8, or CCL8 + Maraviroc. All t -tests were two-tailed. Mean ± SEM. * p < 0.05, ** p < 0.01; *** p < 0.001; ns, no significance. Original western blots are presented in . Scale bar: 100 μm.

Article Snippet: Recombinant human CCL8 (rCCL8) protein, macrophage colony-stimulating factor (MCSF), and Maraviroc (selective antagonist of CCR5) were purchased from MCE.

Techniques: Western Blot, Colony Assay, Immunohistochemistry, Two Tailed Test

Journal: Cancers

Article Title: Lactate-Induced CCL8 in Tumor-Associated Macrophages Accelerates the Progression of Colorectal Cancer through the CCL8/CCR5/mTORC1 Axis

doi: 10.3390/cancers15245795

Figure Lengend Snippet: ( A – C ) The alterations of the mTOR/4EBP1/70S6K signaling pathway in RKO cells, after treatment with CCL8, Maraviroc, or a series of combinations of CCL8 and Maraviroc for 24 h. ( D ) The alterations in the mTOR/4EBP1/70S6K signaling pathway in RKO cells before and after CCR5 knockdown, treated with or without CCL8 for 24 h. Original western blots are presented in .

Article Snippet: Recombinant human CCL8 (rCCL8) protein, macrophage colony-stimulating factor (MCSF), and Maraviroc (selective antagonist of CCR5) were purchased from MCE.

Techniques: Knockdown, Western Blot

Journal: Cancers

Article Title: Lactate-Induced CCL8 in Tumor-Associated Macrophages Accelerates the Progression of Colorectal Cancer through the CCL8/CCR5/mTORC1 Axis

doi: 10.3390/cancers15245795

Figure Lengend Snippet: Schematic model showing the tumor-progression-promoting interaction between CRC cells and TAMs. Tumor-associated macrophages (TAMs) are more likely to adopt an M2-type polarization in response to the high lactate level in the tumor microenvironment. This leads to an increase in the secretion of CCL8 by polarized TAMs, which binds to the CCR5 receptor on tumor cells and promotes their proliferation and metastasis via the mTOR/70S6K/4EBP1 signaling pathway.

Article Snippet: Recombinant human CCL8 (rCCL8) protein, macrophage colony-stimulating factor (MCSF), and Maraviroc (selective antagonist of CCR5) were purchased from MCE.

Techniques:

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: