recombinant human rantes (ccl5)  (PeproTech)

Journal: Biomedicines

Article Title: The Chemokine (C-C Motif) Receptor 1 Antagonist BX471 Improves Fluid Resuscitation in Rat Models of Hemorrhagic Shock

doi: 10.3390/biomedicines13051241

Figure Lengend Snippet: Serum CCR1 ligand concentrations (pg/mg) at baseline (t = 0 min) and at the end of the experiment (t = 210 min). Light grey bars: vehicle treatment, n = 7. Dark grey bars: 0.5 μmol/kg BX471 treatment, n = 7. ( A ) CCL3, ( B ) CCL4, ( C ) CCL5, and ( D ) CCL7. *: p < 0.05 vs. baseline (t = 0 min).

Article Snippet: Measurements of Chemokine Concentrations: Serum levels of CCL3, CCL4, CCL5, and CCL7 were measured with commercially available rat enzyme-linked immunosorbent assays (ELISA, Boster Bio, Pleasanton, CA, USA) according to the manufacturer’s instructions.

Techniques:

Journal: Frontiers in Immunology

Article Title: NOX4 modulates breast cancer progression through cancer cell metabolic reprogramming and CD8 + T cell antitumor activity

doi: 10.3389/fimmu.2025.1534936

Figure Lengend Snippet: NOX4 enhanced the CD8 + T cells mediate antitumor effect. (A) TIMER2.0 was used to analysis the correlation between NOX4 expression levels and CD8 + T cell infiltration, Scatter plot showing the correlation between NOX4 expression levels and CD8 + T cell infiltration in breast cancer (BRCA) samples (n=1100). Rho=0.346, p=2.57e-26. (B) TISIDB data: Scatter plot showing the correlation between NOX4 expression and central memory CD8 + T cell (Tcm-CD8) infiltration, effector memory CD8+ T cell (Tem-CD8) in BRCA samples (n=1100). Rho=0.44, p<2.2e-16; Rho=0.171, p<1.24e-6. (C) Representative flow cytometry plots showing the percentage of CD8 + T cells within the CD45 + population in EO771 tumors from NOX4 wild-type (WT) and NOX4 knockout (KO) mice. Bar graph quantifying the percentage of CD8 + cells, showing a significant reduction in CD8 + T cell infiltration in NOX4 KO tumors ***p<0.001. n=6 per group. (D) Representative flow cytometry plots showing IFN-γ and Granzyme B expression in CD8 + T cells from EO771 tumors of NOX4 WT and NOX4 KO mice. Bar graphs quantifying the percentage of CD8 + T cells expressing IFN-γ and Granzyme B, ***p<0.001. n=7 per group. (E) Representative flow cytometry plots showing the percentage of DC cells within the EO771 tumors from NOX4 wild-type (WT) and NOX4 knockout (KO) mice. Bar graph quantifying the percentage of DC cells, **p<0.01. n=7 per group. (F) TISIDB data: Scatter plot showing the correlation between NOX4 expression and CCL11 expression in BRCA samples (n=1100). A significant positive correlation is observed. Rho=0.359, p<2.2e-16. Bar graph showing the concentration of CCL11, CCL5 in the tumor microenvironment, *p<0.05. Data are presented as mean ± SEM from 3 independent experiments. Statistical significance was determined using two-tailed Student’s t-test.

Article Snippet: The quantity of CCL11, CCL5 (Bosterbio) were determined in tumor tissue using ELISA kits according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Knock-Out, Concentration Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

doi: 10.1038/s41598-024-75327-y

Figure Lengend Snippet: Purification of recombinant IH-CCL5 using Shuffle lysY cells. ( a ) Workflow of IH-CCL5 purification, figure generated using BioRender. ( b ) His 6 -SUMO-CCL5 construct representation. ( c ) SDS-PAGE analysis of SUMO-CCL5 post Ni 2+ purification. Lane representation follows lane 1: non-induced total protein (TP), lane 2: non-induced soluble (S), lane 3: induced TP, lane 4: induced S, lane 5: induced insoluble (Ins), lane 6: pre-column, lane 7: His-column flow though, lane 8: His-column wash, lane 9–13: elution fractions F4,6,8,10 and 12. ( d ) SUMO cleavage of His 6 -SUMO-CCL5 construct representation. ( e ) SDS-PAGE analysis post nickel column purification of SUMO cleaved CCL5. Lane 1: His 6 -SUMO-CCL5 pre-cleavage (PrC), lane 2: cleaved CCL5 (C), lane 3: column flow-through (FT) and lane 4: column wash (W).

Article Snippet: Commercial recombinant human RANTES (CCL5) produced in E. Coli (with endotoxin level guaranteed ≤ 1 EU/ug) was purchased from Peprotech.

Techniques: Purification, Recombinant, Generated, Construct, SDS Page, Nickel Column

Journal: Scientific Reports

Article Title: New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

doi: 10.1038/s41598-024-75327-y

Figure Lengend Snippet: CCL5-mediated activation of CCR5. Ligand binding to CCR5 induces receptor phosphorylation, intracellular signals and calcium flux before removal of ß-arrestin (ß-arr.)-bound surface receptors by internalisation leading to CCR5 downmodulation.

Article Snippet: Commercial recombinant human RANTES (CCL5) produced in E. Coli (with endotoxin level guaranteed ≤ 1 EU/ug) was purchased from Peprotech.

Techniques: Activation Assay, Ligand Binding Assay, Phospho-proteomics

Journal: Scientific Reports

Article Title: New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

doi: 10.1038/s41598-024-75327-y

Figure Lengend Snippet: IH-CCL5 binding induces CCR5 phosphorylation on CHO-CCR5 cells. IH-CCL5 binding and kinetics of CCR5 phosphorylation using IH-CCL5 over the course of 30 min. ( a ) Flow cytometry analysis of IH-CCL5 binding to CCR5, assessed by loss of anti-CCR5 2D7 signal. Histograms for cells in medium (grey filled) and 100 nM IH-CCL5 (blue solid line) overlayed with no 2D7 (grey dotted line). Bar chart displaying change in specific MFI between medium and IH-CCL5 treated cells. Data shown from a representative experiment. Data analysed with t-test *** P < 0.0002. ( b ) CHO-CCR5 immunoblot after 100 nM IH-CCL5 stimulation for up to 60 min. Anti-CCR5 mAb MC5 (1 µg/mL) was used to detect CCR5 and histone-3 (H3) as a loading control (full-length blot in supplementary Fig. 4). ( c ) Fold change in E11/19-APC signal (phospho-FLOW) over 30 min CCR5 stimulation with 100 nM IH-CCL5 ( n = 3). **** P ≤ 0.0001 two-way ANOVA. Graph symbols medium (open square), IH-CCL5 (filled square) and IH-CCL5/MVC (filled triangle).

Article Snippet: Commercial recombinant human RANTES (CCL5) produced in E. Coli (with endotoxin level guaranteed ≤ 1 EU/ug) was purchased from Peprotech.

Techniques: Binding Assay, Phospho-proteomics, Flow Cytometry, Western Blot, Control

Journal: Scientific Reports

Article Title: New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

doi: 10.1038/s41598-024-75327-y

Figure Lengend Snippet: In-house CCL5 induced downstream CCR5 signalling activity. Chemokine binding activates downstream signalling that facilitates the release of calcium and cell migration. ( a ) Flow cytometry based experimental workflow for measuring calcium release upon chemokine stimulation. Cells were loaded with Fluo-8 AM (calcium reporter dye) and stimulated with 100 nM chemokine +/- TAK-779 and calcium release was measured using CyAn flow cytometer (FITC). Arrows on the graphs show time at which ( b ) 100 nM commercial CCL5 or ( c ) 100 nM IH-CCL5 were added. Results are from a representative experiment. CCL5 induced activation of CCR5 induces downstream signalling, which is involved in cell migration. ( d ) IH-CCL5 mediated CHO-CCR5 migration was assed using a Transwell migration assay with a 12 μm polycarbonate membrane pore size. ( e ) CHO-CCR5 cells were stimulated with 10 nM IH-CCL5 with or without 800 nM TAK-779. Cell migration was determined as the number of cells that migrated through the membrane filter into lower chamber ( n = 3). One-way ANOVA statistical analysis with Bonferroni test was performed on the data **** P < 0.0001.

Article Snippet: Commercial recombinant human RANTES (CCL5) produced in E. Coli (with endotoxin level guaranteed ≤ 1 EU/ug) was purchased from Peprotech.

Techniques: Activity Assay, Binding Assay, Migration, Flow Cytometry, Activation Assay, Transwell Migration Assay, Membrane, Pore Size

Journal: Scientific Reports

Article Title: New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

doi: 10.1038/s41598-024-75327-y

Figure Lengend Snippet: IH-CCL5 induced CCR5 downmodulation and internalisation. CCR5 downmodulation was assessed through the loss of anti-CCR5 MC5 binding at 37 o C. ( a , b ) Flow cytometry histogram overlays for MC5 (detected with an anti-mouse A647; APC channel) in different conditions (Medium – Solid grey line with grey fill, 100 nM CCL5 – Blue solid line, 100 nM CCL5 + 800 nM TAK-779 – Red solid line and isotype control – Grey dotted line). ( c ) Change in specific MFI following treatment with the indicated recombinant CCL5 alone (black bars) or with TAK-779 pre-treatment (Grey bars) compared to untreated cells ( n = 3). ** P ≤ 0.0021 and **** P ≤ 0.0001 One-way ANOVA with Bonferroni test. ( d ) Comparison of CCR5 downmodulation induced by IH-CCL5 and commercial CCL5 for CHO-CCR5 cells treated with 100 nM chemokine 1 h at 37 o C ( n = 3). T-test showed no significant difference (ns). ( e ) IH-CCL5 induced CCR5 internalisation using immunofluorescence microscopy. CHO-CCR5 cells were prelabelled with MC5 (5 µg/mL) followed by 100 nM chemokine treatment 0.5 h 37 o C. Secondary anti-mouse A568 4 µg/mL (red) and cells mounted in mowiol containing DAPI (blue). Confocal microscopy images (scale bar at 20 μm) analysed on ImageJ.

Article Snippet: Commercial recombinant human RANTES (CCL5) produced in E. Coli (with endotoxin level guaranteed ≤ 1 EU/ug) was purchased from Peprotech.

Techniques: Binding Assay, Flow Cytometry, Control, Recombinant, Comparison, Immunofluorescence, Microscopy, Confocal Microscopy

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: Recombinant Human CCL5/RANTES Protein , R&D Systems , Cat. #278-RN-010.

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: 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: Recombinant Human CCL5/RANTES Protein , R&D Systems , Cat. #278-RN-010.

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: Recombinant Human CCL5/RANTES Protein , R&D Systems , Cat. #278-RN-010.

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: Recombinant Human CCL5/RANTES Protein , R&D Systems , Cat. #278-RN-010.

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: Recombinant Human CCL5/RANTES Protein , R&D Systems , Cat. #278-RN-010.

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