Journal: International Journal of Molecular Sciences

Article Title: Engagement of the CXCL12–CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis

doi: 10.3390/ijms23020867

Figure Lengend Snippet: Expression and release of CXCL12 by SMCs and EPCs. ( A ) Analysis of CXLC12 release using ELISA. Cell supernatants from monocultured SMCs and EPCs, treated as indicated, were collected 24 h after cultivation. * p < 0.05 vs. untreated cells; n = 6. ( B ) Real-time RT-PCR analysis of CXCL12 expression SMCs and EPCs treated as indicated. Results were normalized to CXCL12 expression in SMCs. * p < 0.05 vs. untreated cells; n = 5. ( C ) Detection of CXCL12 in MVs derived from monocultured SMCs and EPCs. Isolated MVs were lysed in RIPA buffer and CXCL12 levels were determined using ELISA. * p < 0.05 vs. non-injured SMCs, # p < 0.05 vs. SMC-MV; n = 5. ( D ) Enumeration of MVs in the supernatant of EPCs, non-injured SMCs and injured SMCs using flow cytometry with calibrated microbeads. * p < 0.05 vs. non-injured SMCs; n = 4. ( E ) Evaluation of the effect of EPC-SMC co-cultivation and engagement of CXCR4 on the release of CXCL12. Supernatants from monocultured SMCs, monocultured EPCs and EPCs co-cultured with SMCs, each in the presence or absence of a blocking CXCR4 Ab, were analyzed for CXCL12 concentration using ELISA. * p < 0.05 vs. SMCs, # p < 0.05 vs. EPC-SMC co-culture in the absence of anti-CXCR4; n = 5. ( F ) Real-time RT-PCR analysis of CXCL12 expression to test the impact of EPC-SMC co-cultivation and involvement of CXCR4. CXCL12 transcripts were determined in SMCs, EPCs and EPC-SMC co-cultures in the presence or absence of a blocking CXCR4 Ab. * p < 0.05 vs. SMCs, # p < 0.05 vs. EPC-SMC co-culture in the absence of anti-CXCR4; n = 5. ( G ) Real-time RT-PCR analysis of CXCL12 expression in SMCs treated with various doses of rCXCL12, CM-EPC or EPC-MV in the presence or absence of an anti-CXCR4 Ab. * p < 0.05 vs. untreated SMCs (control), # p < 0.05 vs. respective treatment in the absence of anti-CXCR4; n = 5. ( H ) Adhesion of EPCs to SMCs under flow conditions in vitro. EPCs pretreated with/without an anti-CXCR4 Ab were perfused in a parallel flow chamber and the number of cells EPCs adherent to the SMC monolayer was determined and expressed as adherent cells per 1 mm². For some experiments, the SMC monolayer was wounded by a linear scratch before perfusion of EPCs. * p < 0.05 vs. untreated and non-scratched SMCs (control), # p < 0.05 vs. respective treatment in the absence of anti-CXCR4; n = 4 to 6.

Article Snippet: In brief, SMCs or HUVECs rendered quiescent by serum starvation for 24 h were seeded in 96-well plates (~50% confluent cells) and subsequently treated with EPCs (5 × 10 4 ), 50 μL CM-EPC, 50 μL CM-EPC/SMC, EPC-MV, recombinant human PDGF-BB (20 ng/mL, R&D Systems) or recombinant human CXCL12 (rCXCL12; 1 to 50 ng/mL, R&D Systems) as indicated.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Derivative Assay, Isolation, Flow Cytometry, Cell Culture, Blocking Assay, Concentration Assay, Co-Culture Assay, Control, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: Engagement of the CXCL12–CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis

doi: 10.3390/ijms23020867

Figure Lengend Snippet: Higher concentration of CXCL12 induce proliferation of SMCs via CXCR4. ( A ) Flow cytometry-based cell cycle analysis of SMCs treated for 24 h with EPCs in the presence or absence of anti-CXCR4 and anti-CXCL12 Abs as indicated. * p < 0.05 vs. untreated SMCs (control); n = 5. ( B – D ) Analysis of SMCs in S phase as determined 24 h after treatment as indicated. * p < 0.05 vs. untreated SMCs (control), # p < 0.05 vs. EPC treated SMCs in the absence of blocking Abs, § p < 0.05 vs. rCXCL12 50 ng/mL treated SMCs; n = 4 to 6. ( E ) Annexin V-FITC/PI staining with subsequent flow cytometry analysis to determine the rate of apoptotic SMCs treated as indicated. * p < 0.05 vs. untreated SMCs (control) for respective early and late apoptosis, # p < 0.05 vs. EPC-treated SMCs for late apoptosis; n = 5.

Article Snippet: In brief, SMCs or HUVECs rendered quiescent by serum starvation for 24 h were seeded in 96-well plates (~50% confluent cells) and subsequently treated with EPCs (5 × 10 4 ), 50 μL CM-EPC, 50 μL CM-EPC/SMC, EPC-MV, recombinant human PDGF-BB (20 ng/mL, R&D Systems) or recombinant human CXCL12 (rCXCL12; 1 to 50 ng/mL, R&D Systems) as indicated.

Techniques: Concentration Assay, Flow Cytometry, Cell Cycle Assay, Control, Blocking Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Engagement of the CXCL12–CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis

doi: 10.3390/ijms23020867

Figure Lengend Snippet: EPCs stimulate migration of SMCs via CXCL12-CXCR4. ( A , B ) Transmigration of SMCs as analyzed in transwell chamber experiments with 8 μm pores and expressed as percentage of control. The bottom chamber contained migration medium (DMEM plus 0.5% FBS) supplemented with various doses of rCXCL12, EPCs or their secretory products in the absence or presence of blocking Abs as indicated. * p < 0.05 vs. control, # p < 0.05 vs. respective treatment in the absence of blocking Abs; n = 6. ( C , D ) SMC scratch assay. Subconfluent monolayers of SMCs, treated as indicated, were wounded linearly, and the area of the wound subsequently recovered by migrated SMCs was expressed as a percentage of the initial wound area. Representative photomicrographs ( C ) and quantified data ( D ) are shown. * p < 0.05 vs. untreated SMCs (control), # p < 0.05 vs. respective treatment in the absence of blocking Abs; n = 6.

Article Snippet: In brief, SMCs or HUVECs rendered quiescent by serum starvation for 24 h were seeded in 96-well plates (~50% confluent cells) and subsequently treated with EPCs (5 × 10 4 ), 50 μL CM-EPC, 50 μL CM-EPC/SMC, EPC-MV, recombinant human PDGF-BB (20 ng/mL, R&D Systems) or recombinant human CXCL12 (rCXCL12; 1 to 50 ng/mL, R&D Systems) as indicated.

Techniques: Migration, Transmigration Assay, Control, Blocking Assay, Wound Healing Assay

Journal: International Journal of Molecular Sciences

Article Title: Engagement of the CXCL12–CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis

doi: 10.3390/ijms23020867

Figure Lengend Snippet: Engagement of CXCL12–CXCR4 in proliferation and migration of endothelial cells. ( A ) Flow-cytometry-based cell cycle analysis of HUVECs treated for 24 h as indicated. * p < 0.05 vs. untreated HUVECs (control), # p < 0.05 vs. respective treatment in the absence of blocking Abs; n = 5. ( B ) Transmigration of HUVECs as analyzed in transwell chamber experiments and expressed as percentage of control. The bottom chamber contained migration medium (DMEM plus 0.5% FBS) supplemented with/without various doses of rCXCL12, CM-EPC or CM-EPC/SMC in the absence or presence of blocking Abs as indicated. * p < 0.05 vs. control, # p < 0.05 vs. respective treatment in the absence of blocking Abs; n = 6. ( C ) HUVEC scratch assay. Monolayers of HUVECs, treated as indicated, were wounded linearly, and the area of the wound subsequently recovered by migrated HUVECs was expressed as a percentage of the initial wound area. * p < 0.05 vs. untreated HUVECs (control), # p < 0.05 vs. respective treatment in the absence of blocking Abs; n = 6.

Article Snippet: In brief, SMCs or HUVECs rendered quiescent by serum starvation for 24 h were seeded in 96-well plates (~50% confluent cells) and subsequently treated with EPCs (5 × 10 4 ), 50 μL CM-EPC, 50 μL CM-EPC/SMC, EPC-MV, recombinant human PDGF-BB (20 ng/mL, R&D Systems) or recombinant human CXCL12 (rCXCL12; 1 to 50 ng/mL, R&D Systems) as indicated.

Techniques: Migration, Flow Cytometry, Cell Cycle Assay, Control, Blocking Assay, Transmigration Assay, Wound Healing Assay

Journal: International Journal of Molecular Sciences

Article Title: Engagement of the CXCL12–CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis

doi: 10.3390/ijms23020867

Figure Lengend Snippet: CXCL12–CXCR4-dependent control of SMC phenotype. ( A – E ) Analysis of EPC-mediated modulation of SMC phenotype and the involvement of the CXCL12–CXCR4 axis. SMCs were treated as indicated for 48 h and presence of SMA and Calponin was measured using flow cytometry. Data are expressed as mean fluorescence intensity (MFI) in % normalized to untreated SMCs (control). ( A – C ) * p < 0.05 vs. control and # p < 0.05 vs. SMCs treated with rCXCL2 50 ng/mL, ( D ) * p < 0.05 vs. SMCs co-cultured with EPCs, ( E ) * p < 0.05 vs. control and # p < 0.05 vs. the respective treatment of SMCs with CM-EPC or EPC-MV in the absence of anti-PDGFRß; n = 4 to 6. ( F ) Comparison of CXCL12 and PDGF-BB released by monocultured EPCs, monocultured SMCs and EPC-SMC co-cultures. Secreted paracrine factors present in the supernatant of the respective cultures were assessed using ELISA; n = 5.

Article Snippet: In brief, SMCs or HUVECs rendered quiescent by serum starvation for 24 h were seeded in 96-well plates (~50% confluent cells) and subsequently treated with EPCs (5 × 10 4 ), 50 μL CM-EPC, 50 μL CM-EPC/SMC, EPC-MV, recombinant human PDGF-BB (20 ng/mL, R&D Systems) or recombinant human CXCL12 (rCXCL12; 1 to 50 ng/mL, R&D Systems) as indicated.

Techniques: Control, Flow Cytometry, Fluorescence, Cell Culture, Comparison, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Engagement of the CXCL12–CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis

doi: 10.3390/ijms23020867

Figure Lengend Snippet: Engagement of CXCL12–CXCR4 in EPC-mediated protection of cholesterol-induced phenotype switch. SMCs were treated as indicated for various time periods and presence of stated phenotype markers was determined using flow cytometry. Data are expressed as MFI in % normalized to untreated SMCs (control). ( A , B ) Assessment of the transformative potential of CXCL12 in the presence of SMC loading with Chol:MßCD complexes (Chol). SMCs were treated as indicated for 48 h. Anti-CXCR4 Ab and/or rCXCL12 were added to SMCs simultaneously with Chol:MßCD. * p < 0.05 vs. untreated SMCs (control), # p < 0.05 vs. SMCs treated with Chol, § p < 0.05 vs. SMCs treated with rCXCL2 50 ng/mL plus Chol:MßCD; n = 5. ( C , D , F , G , H ) Time course of SMA and CD68 expression by SMCs treated as indicated for various time periods. SMCs loaded with Chol:MßCD were either continuously co-cultured with rCXCL12 or EPCs for up to 96 h or were primary treated with Chol:MßCD for 48 h and only then subsequently exposed to rCXCL12 or EPCs for another 48 h (post-exposure); n = 4 to 6. ( E ) Analysis of CXCL12–CXCR4-dependent and EPC-mediated protection of cholesterol-induced phenotype switch after 48 h. * p < 0.05 vs. SMCs treated with Chol:MßCD, # p < 0.05 vs. SMCs co-cultured with EPCs and treated with Chol:MßCD plus EPCs (Chol.-EPC); n = 4.

Article Snippet: In brief, SMCs or HUVECs rendered quiescent by serum starvation for 24 h were seeded in 96-well plates (~50% confluent cells) and subsequently treated with EPCs (5 × 10 4 ), 50 μL CM-EPC, 50 μL CM-EPC/SMC, EPC-MV, recombinant human PDGF-BB (20 ng/mL, R&D Systems) or recombinant human CXCL12 (rCXCL12; 1 to 50 ng/mL, R&D Systems) as indicated.

Techniques: Flow Cytometry, Control, Expressing, Cell Culture

Journal: International immunology

Article Title: The effects of microenvironment and internal programming on plasma cell survival.

doi: 10.1093/intimm/dxm051

Figure Lengend Snippet: Fig. 4. Effects of the addition rCXCL12 on PC survival. (A) Evidence that bone marrow PCs express CXCR4, the receptor for CXCL12. Bone marrow cells from Cxcr4-EGFP mice were stained with anti-CD138 antibody and analyzed by flow cytometry. Cells were gated based on parameters used for sorting PCs. CD138 bright cells were assessed for GFP indicating expression of CXCR4. The mean fluorescence intensity of cells negative for GFP is 143. (B and C) Purified bone marrow PCs were cultured with varying concentrations of rCXCL12 (B) or rIL-6 (1 ng ml1) or rIL-6 + rCXCL12 (10 ng ml1) (C). Data shown are the averages of two independent experiments 6 SEM as determined by ELISA. *P < 0.05 as compared with untreated PCs as determined by a student’s t test. In both experiments, the data from cells receiving IL-6 or IL-6 + CXCL12 was significant as compared with untreated PCs; there was no statistical difference between cells receiving IL-6 versus IL-6 + CXCL12.

Article Snippet: Recombinant CXCL12 and neutralizing anti-CXCL12 antibody Bone marrow PCs were isolated as described above and cultured in round-bottom 96-well plates (2500 PCs per well; four wells per treatment group) in the presence of varying concentrations of recombinant mouse CXCL12 (R&D Systems, Minneapolis, MN, USA) for 7 days.

Techniques: Staining, Cytometry, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: The effects of microenvironment and internal programming on plasma cell survival.

doi: 10.1093/intimm/dxm051

Figure Lengend Snippet: Fig. 5. Effects of neutralizing CXCL12 antibody or rCXCL12 on PC/ stromal cell co-cultures. (A) Bone marrow PCs were co-cultured with bone marrow-purified stromal cells in the presence or absence of neutralizing anti-CXCL12 antibody (10 lg ml1) or isotype control antibody (10 lg ml1). Day 7 supernatants were collected and antibody concentrations determined by ELISA. (B) Bone marrow PCs were co-cultured with stromal cells purified from bone marrow, spleen and lymph node cultures in the presence or absence of rCXCL12 (10 ng ml1). Day 7 supernatants were collected and antibody concen- trations determined by ELISA. Data are expressed as the percentage increase in antibody secretion following addition of rCXCL12 as compared with PCs co-cultured with organ-specific stromal cells alone.

Article Snippet: Recombinant CXCL12 and neutralizing anti-CXCL12 antibody Bone marrow PCs were isolated as described above and cultured in round-bottom 96-well plates (2500 PCs per well; four wells per treatment group) in the presence of varying concentrations of recombinant mouse CXCL12 (R&D Systems, Minneapolis, MN, USA) for 7 days.

Techniques: Cell Culture, Control, Enzyme-linked Immunosorbent Assay

Journal: Nagoya Journal of Medical Science

Article Title: RHOF PROMOTES MURINE MARGINAL ZONE B CELL DEVELOPMENT

doi:

Figure Lengend Snippet: Generation of RhoF KO mice. A. The targeting vector, the wild-type RhoF allele, the targeted allele before (flox allele) and after Cre-mediated excitation of the loxP franked Neo cassette are schematically represented. Black boxes in the genomic structures represent exon sequences. Exon 2 of RhoF gene and an inserted FRT-Neo selection marker cassette were flanked by loxP sites such that deletion of exon 2 caused a frameshift mutation resulting in the loss of function. A constitutive knock-out was generated after Cre-mediated deletion of exon 2. Correct Cre-mediated excitation of the loxP franked Neo cassette was confirmed by the appearance of a 2.7 kb recombined instead of 3.0 kb targeted fragment in the PCR products.

Article Snippet: In the lower chamber, 600 μl of 10% FBS/RPMI1640 with or without 800 ng/ml of CXCL13 (470-BC-025/CF; R&D systems, Minneapolis, MN, USA) or 200 ng/ml of stromal cell-derived factor-1α (SDF-1α; 460-SD-010/CF; R&D systems) was added.

Techniques: Plasmid Preparation, Selection, Marker, Mutagenesis, Knock-Out, Generated

Journal: Nagoya Journal of Medical Science

Article Title: RHOF PROMOTES MURINE MARGINAL ZONE B CELL DEVELOPMENT

doi:

Figure Lengend Snippet: Generation of RhoF KO mice. B. Western blot analysis of the immune tissues in the wild-type (WT) or RhoF KO mice (7 weeks old, female). RhoF expression was observed in the spleen, thymus, and mesenteric lymph node (MLN) of the WT mice. On the other hand, the expression was absent in the KO mice. RhoF was not expressed in the liver both in WT and RhoF KO mice. α-tubulin was used as a loading control. Thirty micrograms of protein/lane was loaded. Representative figures are shown from one of the two independent experiments.

Article Snippet: In the lower chamber, 600 μl of 10% FBS/RPMI1640 with or without 800 ng/ml of CXCL13 (470-BC-025/CF; R&D systems, Minneapolis, MN, USA) or 200 ng/ml of stromal cell-derived factor-1α (SDF-1α; 460-SD-010/CF; R&D systems) was added.

Techniques: Western Blot, Expressing

Journal: Nagoya Journal of Medical Science

Article Title: RHOF PROMOTES MURINE MARGINAL ZONE B CELL DEVELOPMENT

doi:

Figure Lengend Snippet: Analysis of B cell development in bone marrow and spleen. A. FACS analysis of BM B cells. BM cells from WT and RhoF KO mice (6–7 weeks old, n = 5, male: 40%) were stained with antibodies against CD43, B220, BP1, and CD24 and analyzed by flow cytometry. The number shows the percentage (mean ± standard deviation) of the indicated subpopulation within the parent population; Fraction A (germline pro-B cells), fraction B (DJ-rearranged pro-B cells), fraction C (Early pre-B cells), fraction D (Late pre-B cells), fraction E (newly formed B cells), and fraction F (follicular-type recirculating B cells). The figures are representatives of three independent experiments. B. FACS analysis of B cells from the spleen. Splenocytes from WT and RhoF KO mice (6–7 weeks old, n = 6, male: 33%) were stained with antibodies against CD23, CD21, and IgM and analyzed by flow cytometry. The number shows the percentage (mean ± standard deviation) of the indicated subpopulation within the parent population; T1 (Transitional 1), T2 (Transitional 2), Fo (mature follicular B cells), and MZ (MZ B cells). C. The number of B cell subsets in BM was calculated by multiplying the total number of viable (trypan blue negative) BM cells by the fraction of the target population in viable (7AAD negative) cells. The data are shown as mean ± standard deviation (6–7 weeks old, n = 5, male: 40%). White bars: WT. Black bars: RhoF KO. D. The number of B cell subsets in the spleen was also calculated by multiplying the total number of viable (trypan blue negative) splenocytes by the fraction of the target population in viable (7AAD negative) cells. The data were shown as mean ± standard deviation (6–7 weeks old, n = 6, male: 33%). White bars: WT. Black bars: RhoF KO. *: P < 0.05, WT vs. RhoF KO E. Immunohistostaining of CD169 (Green, MZ metallophilic macrophages) and B220 (Red, B cells) in WT (upper left panel) and RhoF KO (lower left panel) spleen. Scale bars: 100 μm. The widths of the MZ B cell region (B220-positive region outside CD169-positive cells) in the WT and RhoF KO mice are summarized (right panel, n = 32). Four MZ B cell regions per section were captured for four individual sections per mouse, and two mice per group were used for this experiment. Circles: WT. Crosses: RhoF KO. ***: P < 0.0001, WT vs. RhoF KO

Article Snippet: In the lower chamber, 600 μl of 10% FBS/RPMI1640 with or without 800 ng/ml of CXCL13 (470-BC-025/CF; R&D systems, Minneapolis, MN, USA) or 200 ng/ml of stromal cell-derived factor-1α (SDF-1α; 460-SD-010/CF; R&D systems) was added.

Techniques: Staining, Flow Cytometry, Standard Deviation

Journal: Nagoya Journal of Medical Science

Article Title: RHOF PROMOTES MURINE MARGINAL ZONE B CELL DEVELOPMENT

doi:

Figure Lengend Snippet: Immune response and migration of B cells. A. Littermate WT control and RhoF KO mice were immunized with 20 μg of TNP-LPS (200 μl/mouse; WT, 6–8 weeks old, n = 7, male: 86%, KO, 6–9 weeks old, n = 5, male: 80%) or PBS (200 μl/mouse; WT, 6–15 weeks old, n = 9, male: 78%, KO, 6–18 weeks old, n = 9, male: 78%) by injection into the peritoneal cavity and bled at designated time points. Antibody titers of TNP-specific IgM were measured by ELISA. Circles: WT. Crosses: RhoF KO. B. Antibody titers of TNP-specific IgG3 at the indicated time point were measured by ELISA. The samples were same as those for TNP-specific IgM in Figure 5A. Circles: WT. Crosses: RhoF KO. C. Migration assay of MZ B cells. Total splenocytes (10 6 cells/well) were placed in the upper chamber of Transwell, which was set on the lower chamber with 10% FBS/RPMI 1640 medium with or without BLC (800 ng/ml, BLC) or SDF-1α (200 ng/ml, SDF1). In this experiment, FACS analysis (with anti-CD21, IgM, and CD23) was performed using the input cells and cells in the lower chamber after incubation, to calculate the percentage of MZ B cells that migrated into the lower chamber. The data are shown as mean ± standard deviation (11–12 weeks old, n = 4, male: 50%) from two independent experiments. White bars: WT. Black bars: RhoF KO.

Article Snippet: In the lower chamber, 600 μl of 10% FBS/RPMI1640 with or without 800 ng/ml of CXCL13 (470-BC-025/CF; R&D systems, Minneapolis, MN, USA) or 200 ng/ml of stromal cell-derived factor-1α (SDF-1α; 460-SD-010/CF; R&D systems) was added.

Techniques: Migration, Injection, Enzyme-linked Immunosorbent Assay, Incubation, Standard Deviation

Journal: Journal of Korean Medical Science

Article Title: LPS-Induced Migration of Peritoneal B-1 Cells is Associated with Upregulation of CXCR4 and Increased Migratory Sensitivity to CXCL12

doi: 10.3346/jkms.2012.27.1.27

Figure Lengend Snippet: Heightened migratory response of peritoneal B cells obtained from LPS-treated mice in response to CXCL12 or CXCL13. In vitro transwell migration assay was performed with peritoneal B cells isolated from 10 week-old C57BL/6 mice at the indicated time after intraperitioneal injection of LPS. Peritoneal B cells were added to the upper chamber of a transwell plate in the presence of CXCL12 or CXCL13 in the lower chamber. Two hour later, the numbers of B-1a, B-1b, and B-2 cells in the lower chambers were counted. Results represent the percentage of the cell number in the lower chamber over the input number in response to medium alone, CXCL12, or CXCL13. Data were collected from three mice each experimental group. Statistical analyses were performed by Student's t test for comparison between groups of treatment with PBS and LPS (24 and 48 hr) (above each bar) and also between B-1a cells and B-2 cells or B-1b cells (above each drawn line). n.s., differences not statistically significant. * P = 0.01-0.05; † P = 0.001-0.01.

Article Snippet: Mouse recombinant CXCL12, CXCL13, and anti-mouse CXCL12 antibody were purchased from the R&D systems (Minneapolis, MN, USA).

Techniques: In Vitro, Transwell Migration Assay, Isolation, Injection, Comparison

Journal: Journal of Korean Medical Science

Article Title: LPS-Induced Migration of Peritoneal B-1 Cells is Associated with Upregulation of CXCR4 and Increased Migratory Sensitivity to CXCL12

doi: 10.3346/jkms.2012.27.1.27

Figure Lengend Snippet: Increased chemotactic response of LPS-stimulated peritoneal B cells in response to CXCL12 or CXCL13. In vitro migration assay was performed with purified peritoneal B cells stimulated for 24 hr with or without LPS in vitro. Peritoneal B cells were added to the upper chamber of transwell plate in the presence of CXCL12 or CXCL13 in the lower chamber. 2 hr later, the numbers of B-1a, B-1b, and B-2 cells in the lower chambers were counted. Results represent the percentage of the cell number in the lower chamber over the input number in response to medium alone, CXCL12, or CXCL13. Statistical analyses from three mice of each experimental group were performed by Student's t test in comparison with the group of media treatment. n.s., differences not statistically significant. * P = 0.01-0.05; † P = 0.001-0.01.

Article Snippet: Mouse recombinant CXCL12, CXCL13, and anti-mouse CXCL12 antibody were purchased from the R&D systems (Minneapolis, MN, USA).

Techniques: In Vitro, Migration, Purification, Comparison

Journal: Journal of Korean Medical Science

Article Title: LPS-Induced Migration of Peritoneal B-1 Cells is Associated with Upregulation of CXCR4 and Increased Migratory Sensitivity to CXCL12

doi: 10.3346/jkms.2012.27.1.27

Figure Lengend Snippet: The concentration of CXCL12 in the peritoneal fluids from LPS-injected mice. Peritoneal fluids isolated from 10 week-old C57BL/6 mice at the indicated time after intraperitioneal injection of LPS were measured for the CXCL12 concentrations by ELISA. Statistical analysis from three mice of each group was performed by Student's t test in comparison with the concentration of PBS treatment group. n.s., differences not statistically significant.

Article Snippet: Mouse recombinant CXCL12, CXCL13, and anti-mouse CXCL12 antibody were purchased from the R&D systems (Minneapolis, MN, USA).

Techniques: Concentration Assay, Injection, Isolation, Enzyme-linked Immunosorbent Assay, Comparison

Journal: International Journal of Cell Biology

Article Title: CXCL12 Modulates Prostate Cancer Cell Adhesion by Altering the Levels or Activities of β 1-Containing Integrins

doi: 10.1155/2014/981750

Figure Lengend Snippet: Plasma CXCL12 concentrations in the 3 groups analyzed.

Article Snippet: Human recombinant CXCL12, Quantikine human CXCL12/SDF-1 immunoassay kit, and mouse anti-human CXCR4 were from R&D Systems (Minneapolis, USA).

Techniques: Clinical Proteomics, Control

Journal: International Journal of Cell Biology

Article Title: CXCL12 Modulates Prostate Cancer Cell Adhesion by Altering the Levels or Activities of β 1-Containing Integrins

doi: 10.1155/2014/981750

Figure Lengend Snippet: Mean and median plasma CXCL12 concentrations in subgroup H (Gleason score >7 including 4 + 3) were significantly higher than in subgroup L (Gleason score >7 including 4 + 3).

Article Snippet: Human recombinant CXCL12, Quantikine human CXCL12/SDF-1 immunoassay kit, and mouse anti-human CXCR4 were from R&D Systems (Minneapolis, USA).

Techniques: Clinical Proteomics

Journal: International Journal of Cell Biology

Article Title: CXCL12 Modulates Prostate Cancer Cell Adhesion by Altering the Levels or Activities of β 1-Containing Integrins

doi: 10.1155/2014/981750

Figure Lengend Snippet: (a) DU145 cell spreading on COL-I, FN, 50K, and H/120 fragment of FN in the presence of various concentrations of CXCL12 (200 ng/mL). (b) Attachment of DU145 cells on FN and COL-I in the absence and presence of CXCL12 (200 ng/mL). (c) PC3 cell spreading on COL-I, FN, 50K, and H/120 fragment of FN in the presence of various concentrations of CXCL12 (200 ng/mL). (d) PC3 cell attachment on FN and COL-I in the absence and presence of CXCL12 (200 ng/mL). The level of nonspecific binding, determined from these cells attachment to wells coated with BSA alone, was subtracted. Values shown are mean ± standard deviation of triplicate wells.

Article Snippet: Human recombinant CXCL12, Quantikine human CXCL12/SDF-1 immunoassay kit, and mouse anti-human CXCR4 were from R&D Systems (Minneapolis, USA).

Techniques: Cell Attachment Assay, Binding Assay, Standard Deviation

Journal: International Journal of Cell Biology

Article Title: CXCL12 Modulates Prostate Cancer Cell Adhesion by Altering the Levels or Activities of β 1-Containing Integrins

doi: 10.1155/2014/981750

Figure Lengend Snippet: CXCL12 induces focal adhesion disassembly, actin stress fiber rearrangement, and morphological change in DU145 cells seeded on FN. Cells were incubated for 2 hrs, fixed, and double-stained for vinculin and actin. Arrows in the images indicate localization of vinculin at the end of actin bundles in focal adhesion complexes.

Article Snippet: Human recombinant CXCL12, Quantikine human CXCL12/SDF-1 immunoassay kit, and mouse anti-human CXCR4 were from R&D Systems (Minneapolis, USA).

Techniques: Incubation, Staining

Journal: International Journal of Cell Biology

Article Title: CXCL12 Modulates Prostate Cancer Cell Adhesion by Altering the Levels or Activities of β 1-Containing Integrins

doi: 10.1155/2014/981750

Figure Lengend Snippet: CXCL12 induces focal adhesion formation, actin stress fiber rearrangement, and morphological change in PC3 cells seeded on FN. Cells were incubated for 2 hrs, fixed, and double-stained for vinculin and actin. Arrows in the images indicate localization of vinculin at the end of actin bundles in focal adhesion complexes.

Article Snippet: Human recombinant CXCL12, Quantikine human CXCL12/SDF-1 immunoassay kit, and mouse anti-human CXCR4 were from R&D Systems (Minneapolis, USA).

Techniques: Incubation, Staining

Journal: International Journal of Cell Biology

Article Title: CXCL12 Modulates Prostate Cancer Cell Adhesion by Altering the Levels or Activities of β 1-Containing Integrins

doi: 10.1155/2014/981750

Figure Lengend Snippet: PC3 cells, untreated (0) or treated with CXCL12 (200 ng/mL), were lysed and then cell lysates were analyzed by immunoblotting with anti-tyrosine-phosphorylated site-specific antibody against FAK. The total protein was detected by probing the blots with anti-FAK (bottom panel). The blot was performed three times and a representative one is shown.

Article Snippet: Human recombinant CXCL12, Quantikine human CXCL12/SDF-1 immunoassay kit, and mouse anti-human CXCR4 were from R&D Systems (Minneapolis, USA).

Techniques: Western Blot

Journal: International Journal of Cell Biology

Article Title: CXCL12 Modulates Prostate Cancer Cell Adhesion by Altering the Levels or Activities of β 1-Containing Integrins

doi: 10.1155/2014/981750

Figure Lengend Snippet: Flow cytograph analysis of α 4, β 1, and α 5 integrin subunits in untreated (C curve) and 200 ng/mL CXCL12 (T curve) treated cells. The T curves represent the binding of anti-integrin antibody on treated cells, the C curves represent the binding of anti- α 5 integrin antibody on untreated cells, and the mock curves (M) show the corresponding negative control antibody.

Article Snippet: Human recombinant CXCL12, Quantikine human CXCL12/SDF-1 immunoassay kit, and mouse anti-human CXCR4 were from R&D Systems (Minneapolis, USA).

Techniques: Binding Assay, Negative Control

Journal:

Article Title: Stimulation of Chondrocyte Hypertrophy by Chemokine Stromal Cell-Derived Factor 1 in the Chondro-osseous Junction during Endochondral Bone Formation

doi: 10.1016/j.ydbio.2010.02.033

Figure Lengend Snippet: To determine whether SDF-1 can diffuse into cartilage, 17-day-chicken embryonic sternal cartilage was incubated with SDF-1 (100ng/mL) or without SDF-1 for 1h, 3h, and 24h. 10 µm frozen sections were used to detect SDF-1 by immuno-fluorescent staining with mAb against SDF-1. Fluorescence microscopy showed a progressive increase in SDF-1 staining (red color) surrounding chondrocytes during the 24 h time course (A, B,C) compared to control at 24 h (D). Scale bar = 20 µm.

Article Snippet: Before collecting samples for experiment, the cells were stimulated with SDF-1 (100ng/mL; Cat# 351-FS, R&D Systems, Inc. Minneapolis, MN) for 24h or pretreated with AMD3100 for 2 h (5ug/mL; Cat# 155148–31–5, Sigma-Aldrich, St. Louis, MO), a specific inhibitor for CXCR4, before stimulation with SDF-1.

Techniques: Incubation, Staining, Fluorescence, Microscopy, Control

Journal:

Article Title: Stimulation of Chondrocyte Hypertrophy by Chemokine Stromal Cell-Derived Factor 1 in the Chondro-osseous Junction during Endochondral Bone Formation

doi: 10.1016/j.ydbio.2010.02.033

Figure Lengend Snippet: To confirm that SDF-1 induces chondrocyte hypertrophy in growth plates, 12-day-old chicken tibia growth plates were cultured in the presence of SDF-1 (100ng/mL) or in the absence of SDF-1 for 2, 4, and 6 days. 10 µm frozen sections were used to detect Type X collagen expression by immuno-fluorescent staining with mAb against type X collagen. A progressive increase in the size of the hypertrohic growth plate based on Type X collagen staining was seen. The ratio of the length of the hypertrophic zone to that of the total growth plate was calculated at the different time points (B). (* p<0.05). Scale bar = 100 µm.

Article Snippet: Before collecting samples for experiment, the cells were stimulated with SDF-1 (100ng/mL; Cat# 351-FS, R&D Systems, Inc. Minneapolis, MN) for 24h or pretreated with AMD3100 for 2 h (5ug/mL; Cat# 155148–31–5, Sigma-Aldrich, St. Louis, MO), a specific inhibitor for CXCR4, before stimulation with SDF-1.

Techniques: Cell Culture, Expressing, Staining

Journal: Science Advances

Article Title: Hypoxia and matrix viscoelasticity sequentially regulate endothelial progenitor cluster-based vasculogenesis

doi: 10.1126/sciadv.aau7518

Figure Lengend Snippet: ( A ) Intracardiac injection of GFP-ECFCs, followed by injection of SDF-1α loaded nonhypoxic and hypoxic hydrogels into the flank of nu/nu mice. ( B ) Twelve hours after injection, GFP-ECFCs exhibited single-cell spindle (i) or rounded (ii) morphology in nonhypoxic hydrogels. Host cells (GS-IB4 lectin) were also present as isolated, rounded cells. Under hypoxic conditions, GFP-ECFCs were present in clusters (iii and iv), with some clusters containing sprouting cells (iii). Host cells also exhibited cluster morphology under hypoxic conditions (iii and iv). ( C ) Quantification revealed a similar number of cells under both conditions and ( D ) a slight increase in percent area covered by clusters under hypoxic compared to nonhypoxic conditions. ( E ) Clusters were larger under hypoxic than under nonhypoxic conditions. ( F ) Intracardiac injection of GFP-ECFCs followed by injection of SDF-1α loaded hypoxic (ctl) and hypoxic (DPI) hydrogels into the flank of nu/nu mice. ( G ) Twelve hours after encapsulation, GFP-ECFCs were present as both single cells (ii and iii) and clusters (i and iv) under both conditions. ( H ) An increased number of GFP + cells were present in the control group, and ( I ) the percent area covered by clusters was increased in the control versus DPI-treated group. ( J ) Mean cluster sizes between the two groups were not statistically significantly different. n = 6 nu/nu mice per experiment. Graphical data in (C), (D), (H), and (I) are reported as box and whisker plots from minimum to maximum. Graphical data in (E) and (J) are reported as means ± SD, with all points denoted by dots. * P < 0.05.

Article Snippet: ICAM-1 antibody (BBA3), integrin-β2 antibody (AF1730), SDF-1α (350-NS-010), and the Human Protease Proteome Profiler Array Kit (ARY021B) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Injection, Isolation, Encapsulation, Control, Whisker Assay

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 1. Expression of CXCL12 (A) and CXCR4 (B) mRNA in the endometrium during

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques: Expressing

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 2. Localization of CXCL12 (A) and CXCR4 (B) proteins by

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques:

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 3. Immunoblot analysis of CXCL12 proteins in uterine flushings on Day 15 of

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques: Western Blot

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 4. Expression of CXCL12 and CXCR4 in conceptuses from Days 12 and 15 of

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques: Expressing

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 5. Effects of IFNG on CXCL12 and CXCR4 mRNA in endometrial explant

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques:

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 6. Effect of CXCL12 on pTr cell proliferation and migration. (A) RT-PCR

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 7. Effects of CXCL12 on migration of PBMCs and CD4+, CD8+, CD4+CD8+ T

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques: Migration

Journal: Breast Cancer Research : BCR

Article Title: Loss of interferon regulatory factor 5 (IRF5) expression in human ductal carcinoma correlates with disease stage and contributes to metastasis

doi: 10.1186/bcr3053

Figure Lengend Snippet: Genes differentially regulated by IRF5 in MDA-MB-231 cells.

Article Snippet: Briefly, 100 ng/ml human recombinant CXCL12/SDF-1 (R&D Systems, Minneapolis, MN, USA) was added to 600 μl of phenol red-free DMEM medium supplemented with 10% FBS in the lower chamber.

Techniques: Expressing