rantes  (R&D Systems)

Journal: Stem cell research

Article Title: CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

doi: 10.1016/j.scr.2012.11.004

Figure Lengend Snippet: Fig. 3 ADSCs may be affected by endogenous CCL5 signaling. (A) Expression of CCR1 ligands CCL3 and CCL5 in peripheral blood mononuclear cells (BCs), 6 samples of ADSCs (ADSCs 1–6) and skin primary fibroblasts (FBs 1–2) was examined by RT-PCR (CCL3/CCL5 and GAPDH targets were amplified for 40 and 20 cycles, respectively). (B) The level of CCL5 mRNA expression in different samples of ADSCs (I, IV, V, VII, VIII) cultivated for different periods of time (passages p1–p12) was measured using qRT-PCR, normalized with GAPDH expression level and set as 1 in the sample Ip9. The other samples were calculated accordingly. The data are presented as an average mean of one measurement performed in triplicates±SD. Concurrently, concentrations of secreted CCL5 protein (pg/ml) in the conditioning media of the samples I p2, I p9, VIII p2, VII p1, VII p3 and VII p4 were measured by ELISA; NA — not analyzed. (C) CCL5 protein in permeabilized naive or treated with 50 ng/ml of recombinant CCL5 ADSCs (left and right panels, respectively) was examined using CCL5 antibody (red). Nuclear staining was performed using DAPI (blue) (40× magnification). (D) Proliferation and viability of ADSCs cultured in the presence of 25 and 50 ng/ml of CCL5 for 24 and 48 h were tested using ViaLight™plus kit. (E) Three independent lineages of ADSCs were treated with 50 ng/ml of CCL5 for 8, 24, 48 and 72 h, and the level of CCL5 mRNA expression was measured in triplicates, normalized with GAPDH expression level and set as 1 in untreated samples. The data are presented as fold of induction of CCL5 expression in CCL5 treated samples over untreated controls±SD; **pb0.01.

Article Snippet: Cells were exposed to CCR1 antibody CKR-1 (C-20), mouse monoclonal CCL5 capture antibody from CCL5/RANTES DuoSet ELISA Development System (R&D System, Wiesbaden, Germany), or BrdU antibody mAbG3G4 (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA) at dilutions of 1:300, 1:500 and 1:200, respectively, for 2 h at RT.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Recombinant, Staining, Cell Culture

Journal: Stem cell research

Article Title: CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

doi: 10.1016/j.scr.2012.11.004

Figure Lengend Snippet: Fig. 4 ADSCs respond to CCL5. (A) ADSCs were stimulated with 50 ng/ml of CCL5 for 3 h (right panel) and subjected to immunostaining using CCR1 antibody (red) and DAPI (blue). Non-stimulated cells are shown in the left panel. (B) ADSCs were treated with different concentrations of CCL5 for 20 min. The levels of phospho-AKT, phospho-ERK, AKT, ERK and GAPDH proteins were detected by WB. (C) CCR1, CCR3 and CCR5 expression levels in two ADSC samples were measured in triplicates by qRT-PCR, normalized with GAPDH mRNA expression levels and set as 1 in sample V (left panel). ADSCs were stimulated with 50 ng/ml of CCL5 for 10 min or 1 h, the cells were lysed and subjected to WB analysis using phospho-ERK, phospho-AKT, ERK, AKT and GAPDH antibodies. (D) Migration capacity of ADSCs towards 50 ng/ml of CCL5 (left panel) was examined using a modified Boyden chamber. As a control, CCL5-free media were used (right panel). The migratory cells were stained and observed under a light microscope using 20× objective.

Article Snippet: Cells were exposed to CCR1 antibody CKR-1 (C-20), mouse monoclonal CCL5 capture antibody from CCL5/RANTES DuoSet ELISA Development System (R&D System, Wiesbaden, Germany), or BrdU antibody mAbG3G4 (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA) at dilutions of 1:300, 1:500 and 1:200, respectively, for 2 h at RT.

Techniques: Immunostaining, Expressing, Quantitative RT-PCR, Migration, Modification, Control, Staining, Light Microscopy

Journal: Stem cell research

Article Title: CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

doi: 10.1016/j.scr.2012.11.004

Figure Lengend Snippet: Fig. 5 CCL5 induces NFκB transcriptional activity. (A) ADSCs were co-transfected with plasmids encoding firefly luciferase reporter under the control of NFκB dependent promoter and Renilla luciferase reporter under SV40 promoter. The cells were stimulated with CCL5 at indicated concentrations for 24 h. Average firefly luciferase activity obtained from three indepen- dent experiments measured in three replicates were normalized to Renilla luciferase values and presented as a fold of induction±SD over luciferase activity in a non-stimulated ADSC sample (set as 1). (B) ADSCs were stimulated with 50 ng/ml of CCL5 for indicated periods of time and several NFκB target gene mRNA expression levels were analyzed by qRT-PCR. Target gene mRNA levels were measured in triplicates and normalized with GAPDH mRNA expression level. The level of the particular gene expression in the untreated control cells at the same time point was set as 1 (indicated by line). Data are represented as an average mean of fold of induction of the indicated gene expression over non- stimulated control±SD obtained from three independent experi- ments. ((A, B) *pb0.05, **pb0.01.)

Article Snippet: Cells were exposed to CCR1 antibody CKR-1 (C-20), mouse monoclonal CCL5 capture antibody from CCL5/RANTES DuoSet ELISA Development System (R&D System, Wiesbaden, Germany), or BrdU antibody mAbG3G4 (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA) at dilutions of 1:300, 1:500 and 1:200, respectively, for 2 h at RT.

Techniques: Activity Assay, Transfection, Luciferase, Control, Expressing, Quantitative RT-PCR, Gene Expression