Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Systematic Evaluation of the Cellular Innate Immune Response During the Process of Human Atherosclerosis

doi: 10.1161/JAHA.115.002860

Figure Lengend Snippet: Antibodies Used in the Present Study

Article Snippet: CCR7/CD197 , Rabbit (polyclonal), IgG , “M1” Macrophages (used in triple stains) , Tris/EDTA , 9 , 1:300 , Bioss Inc..

Techniques:

Journal: Frontiers in Immunology

Article Title: Targeting LAG-3 and PD-1 to Enhance T Cell Activation by Antigen-Presenting Cells

doi: 10.3389/fimmu.2018.00385

Figure Lengend Snippet: Effect of programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) blockade on IFN-γ secretion of different T cell subpopulations after stimulation with TLR-3-DCs. MACS-enriched CD3 + T cells of 8 healthy donor (HDs) were sorted according to CCR7 and CD45 RA expression (A) . The various T cell subpopulations were cocultured with autologous CMV, EBV, influenza, tetanus (CEFT)-pulsed TLR-3-DCs in the presence or absence of α-PD-1 (B) and α-LAG-3 (C) antibody. IFN-γ secretion was determined by cytometric bead array (CBA) assay, and the ratio between concentration with and without blocking antibody was calculated. All data are presented as box-and-whisker plots, and statistical significance was calculated against a fold change of 1.0. * p < 0.05.

Article Snippet: Immunofluorescent staining of T-cell surface antigens was performed using the following fluorescence-conjugated monoclonal antibodies: CD244 (PE, C1.7; 329507 or APC, C1.7; 329511), PD-1 (Brilliant Violet 421, EH12.7H7; 329919), CD3 (FITC, UCHT1; 300406), CD45RA (Brilliant Violet 421, HI100; 304129; all BioLegend), CD160 (APC, 688327; FAB6700A), TIM-3 (PE, 344823; FAB2365P; both R&D Systems), CD8 (PerCP-eFluor 710, SK1; 8046-0087; eBioscience), CD4 (APC-H7, RPA-T4; 560158; BD Biosciences), LAG-3 (ATTO 647N, 17B4; AG-20B-0012TS AdipoGen), CCR7 (CD197, APC, FR 11-11E8; 130-098-125; Miltenyi Biotec).

Techniques: Activation Assay, Expressing, Concentration Assay, Blocking Assay, Whisker Assay

Journal: Frontiers in Immunology

Article Title: CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

doi: 10.3389/fimmu.2018.01949

Figure Lengend Snippet: CCR7-induced Ca 2+ signaling during entry of Cas9-Hoxb8-DCs transduced with Ca 2+ sensor GCaMP6S into the lymph node. (A) Ex vivo time-lapse imaging of lymph nodes within 5 min after intralymphatic injection of 4 × 10 4 Ccr7 +/+ Hoxb8-DCs (left) or Ccr7 −/− Hoxb8-DCs (right) transduced with Ca 2+ sensor GCaMP6S. White arrowheads indicate cells with changes in GCaMP6S signal intensity, indicating changes in Ca 2+ concentration within the cell. For more details see Supplementary Video . Scale bar represents 15 μm. (B) Changes in GCaMP6S signal intensity for each 5 GCaMP6S + Ccr7 +/+ Cas9-Hoxb8-DCs and GCaMP6S + Ccr7 −/− Cas9-Hoxb8-DCs. Data are represented as a difference in GCaMP6S signal intensity for each time point to the median value for next 3 min. Horizontal dashed lines depict thresholds [defined as a change in a signal intensity >1,000 arbitrary units (a.u.)] used to detect Ca 2+ signals. (C) Pie-charts indicating the percentage of GCaMP6S + Ccr7 +/+ Cas9-Hoxb8-DCs or GCaMP6S + Ccr7 −/− Cas9-Hoxb8-DCs with changes in Ca 2+ signals. There is a significant difference between groups ( p < 0.001, two-tailed Fisher's exact test). (D) Number and (E) average duration of Ca 2+ signals (changes in GCaMP6S intensity) per cell for the tracks with at least one recorded Ca 2+ signal. In (D,E) dots represent individual cells and red line median group value. Asterisk (*) indicates significant difference ( p < 0.05), while ns indicates no significant difference (Mann–Whitney test). Data are representative (A,B) or pool (C–E) of 6 independent experiments with total of 7 lymph nodes per cell type.

Article Snippet: Following antibodies and staining reagents were used in this study: Brilliant Violet 510 anti-mouse I-A/I-E (clone M5/114.15.2), PE-Cy7 anti-mouse CD11c (N418), APC rat IgG2c κ Isotype control (RTK4174), PerCP-Streptavidin, PerCP-Cy5.5 anti-mouse CD8α (53–6.7), PerCP anti-mouse CD4 (RM4-5), APC anti-mouse CD40 (3/23), APC anti-mouse CD80 (16-10A1), APC Armenian Hamster IgG Isotype control (HTK888), FITC anti-mouse MHCII/I-Ab (AF6-120.1), APC rat IgG2b κ Isotype control (RTK4530), PE-Cy7 anti-mouse CD11b (N418) (all from Biolegend), PE anti-mouse CD11b (M1/70), PE anti-mouse TCR Vα2 (B20.1) (all from Invitrogen), eF660 anti-mouse CD11b (M1/70), eF450 anti-mouse CD11b (M1/70), PE anti-mouse F4/80 (BM8), PE anti-rat IgG2a k-Isotype control (BR2a), APC anti-mouse CXCR4 (2B11), Alexa Fluor 488 anti-mouse Lyve-1 (ALY7), APC anti-mouse CD117/cKit (ACKα), PE anti-mouse CD135/Flt3 (A2F10), APC anti-mouse CD11c (N418), PE-Cy7 anti-mouse Ly-6A/E/Sca-1 (D7), biotin anti-mouse CD115/M-CSFR (AF598), APC anti-mouse Ly6C (HK1.4), PE-Cy7 anti-mouse CD45.1 (A20), biotin rat IgG2a κ Isotype control (eBR2a), PE rat IgG2b κ Isotype control (eB149/10H5), APC anti-mouse CD86 (GL1), APC rat IgG2a κ Isotype control (eBR2a), PE anti-mouse CD197 (CCR7) (4B12), APC anti-mouse CD197 (CCR7) (4B12), APC-eF780 anti-mouse Ly-6G/GR-1 (RB6-8C5; all from eBioscience), ATT0647 GFP-Booster (ChromoTek), FITC anti-mouse CD317 (PDCA-1; JF05-1C2.4.1) (Miltenyl Biotec), PE anti-mouse CD11c (HL3) (BD Biosciences), PE-Streptavidin and Cy5 anti-mouse B220 (TIB146; both homemade).

Techniques: Transduction, Ex Vivo, Imaging, Injection, Concentration Assay, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

doi: 10.3389/fimmu.2018.01949

Figure Lengend Snippet: Phenotypic and functional verification of CRISPR/Cas9-mediated knockout of Ccr7 in Hoxb8 cell-derived DCs. (A) Cas9-Hoxb8 cells were transduced with a dTom- and CCR7gRNA-encoding lentivirus. Successfully transduced cells were sorted based on the expression of dTom and subsequently differentiated to mature DCs in the presence of GM-CSF followed by treatment with LPS. Ccr7 +/+ and Ccr7 −/− DCs were analyzed by flow cytometry for their expression of CD80 and CCR7. Data are representative for four independent experiments. (B) Analysis of the composition and frequency of insertions and deletions of Ccr7 −/− Cas9-Hoxb8 cells. R, Pearson correlation coefficient; R 2 describes how strongly the calculated chromatograph of the indel distribution correlates with the Sanger sequencing results of the sample DNA. (C) Chemotactic migration of Ccr7 +/+ and Ccr7 −/− DCs for 2 h toward medium alone or 10, 100, and 200 ng/ml CCL21. Data are pooled from 3 independent experiments with n = 8 in total. Mean + SEM; Kruskal–Wallis and Dunn's multiple comparisons test; ns, not significant; ** p < 0.01. (D) Microscopy of popliteal lymph nodes obtained 4 h after intralymphatic injection of YFP-expressing Ccr7 +/+ DCs and dTom-expressing Ccr7 −/− DCs (5–8 × 10 4 cells in 5 μl PBS; scale bar: 200 μm). (E) Total cell counts and (F) relative distribution of Ccr7 +/+ and Ccr7 −/− DCs 4 h after intralymphatic injection into popliteal LNs of B6 mice. (G) Migration distance from the subcapsular sinus (SCS) for the Ccr7 +/+ and Ccr7 −/− DCs that entered LN parenchyma. Dots represent cell number per LN section (E) or individual cells (G) . Data are representative for (D) or pooled from (E–G) two independent experiments with a total of 7 lymph nodes analyzed. Error bars, SD; red bars, median; Mann–Whitney test; * p < 0.05; **** p < 0.0001.

Article Snippet: Following antibodies and staining reagents were used in this study: Brilliant Violet 510 anti-mouse I-A/I-E (clone M5/114.15.2), PE-Cy7 anti-mouse CD11c (N418), APC rat IgG2c κ Isotype control (RTK4174), PerCP-Streptavidin, PerCP-Cy5.5 anti-mouse CD8α (53–6.7), PerCP anti-mouse CD4 (RM4-5), APC anti-mouse CD40 (3/23), APC anti-mouse CD80 (16-10A1), APC Armenian Hamster IgG Isotype control (HTK888), FITC anti-mouse MHCII/I-Ab (AF6-120.1), APC rat IgG2b κ Isotype control (RTK4530), PE-Cy7 anti-mouse CD11b (N418) (all from Biolegend), PE anti-mouse CD11b (M1/70), PE anti-mouse TCR Vα2 (B20.1) (all from Invitrogen), eF660 anti-mouse CD11b (M1/70), eF450 anti-mouse CD11b (M1/70), PE anti-mouse F4/80 (BM8), PE anti-rat IgG2a k-Isotype control (BR2a), APC anti-mouse CXCR4 (2B11), Alexa Fluor 488 anti-mouse Lyve-1 (ALY7), APC anti-mouse CD117/cKit (ACKα), PE anti-mouse CD135/Flt3 (A2F10), APC anti-mouse CD11c (N418), PE-Cy7 anti-mouse Ly-6A/E/Sca-1 (D7), biotin anti-mouse CD115/M-CSFR (AF598), APC anti-mouse Ly6C (HK1.4), PE-Cy7 anti-mouse CD45.1 (A20), biotin rat IgG2a κ Isotype control (eBR2a), PE rat IgG2b κ Isotype control (eB149/10H5), APC anti-mouse CD86 (GL1), APC rat IgG2a κ Isotype control (eBR2a), PE anti-mouse CD197 (CCR7) (4B12), APC anti-mouse CD197 (CCR7) (4B12), APC-eF780 anti-mouse Ly-6G/GR-1 (RB6-8C5; all from eBioscience), ATT0647 GFP-Booster (ChromoTek), FITC anti-mouse CD317 (PDCA-1; JF05-1C2.4.1) (Miltenyl Biotec), PE anti-mouse CD11c (HL3) (BD Biosciences), PE-Streptavidin and Cy5 anti-mouse B220 (TIB146; both homemade).

Techniques: Functional Assay, CRISPR, Knock-Out, Derivative Assay, Transduction, Expressing, Flow Cytometry, Sequencing, Migration, Microscopy, Injection, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

doi: 10.3389/fimmu.2018.01949

Figure Lengend Snippet: The unlimited proliferative capacity of Cas9-Hoxb8 cells allows the consecutive knockout of multiple genes. Ccr7 −/− Cas9-Hoxb8 cells were transduced with a Cerulean- and CXCR4-gRNA-encoding lentivirus. Transduced cells as well as control Cas9-Hoxb8 cells were subsequently differentiated to mature DCs in the presence of GM-CSF followed by the treatment with LPS. DCs were analyzed by flow cytometry for their expression of CD80, CCR7, and CXCR4. Gray curves depict isotype controls. Data are representative of two independent experiments.

Article Snippet: Following antibodies and staining reagents were used in this study: Brilliant Violet 510 anti-mouse I-A/I-E (clone M5/114.15.2), PE-Cy7 anti-mouse CD11c (N418), APC rat IgG2c κ Isotype control (RTK4174), PerCP-Streptavidin, PerCP-Cy5.5 anti-mouse CD8α (53–6.7), PerCP anti-mouse CD4 (RM4-5), APC anti-mouse CD40 (3/23), APC anti-mouse CD80 (16-10A1), APC Armenian Hamster IgG Isotype control (HTK888), FITC anti-mouse MHCII/I-Ab (AF6-120.1), APC rat IgG2b κ Isotype control (RTK4530), PE-Cy7 anti-mouse CD11b (N418) (all from Biolegend), PE anti-mouse CD11b (M1/70), PE anti-mouse TCR Vα2 (B20.1) (all from Invitrogen), eF660 anti-mouse CD11b (M1/70), eF450 anti-mouse CD11b (M1/70), PE anti-mouse F4/80 (BM8), PE anti-rat IgG2a k-Isotype control (BR2a), APC anti-mouse CXCR4 (2B11), Alexa Fluor 488 anti-mouse Lyve-1 (ALY7), APC anti-mouse CD117/cKit (ACKα), PE anti-mouse CD135/Flt3 (A2F10), APC anti-mouse CD11c (N418), PE-Cy7 anti-mouse Ly-6A/E/Sca-1 (D7), biotin anti-mouse CD115/M-CSFR (AF598), APC anti-mouse Ly6C (HK1.4), PE-Cy7 anti-mouse CD45.1 (A20), biotin rat IgG2a κ Isotype control (eBR2a), PE rat IgG2b κ Isotype control (eB149/10H5), APC anti-mouse CD86 (GL1), APC rat IgG2a κ Isotype control (eBR2a), PE anti-mouse CD197 (CCR7) (4B12), APC anti-mouse CD197 (CCR7) (4B12), APC-eF780 anti-mouse Ly-6G/GR-1 (RB6-8C5; all from eBioscience), ATT0647 GFP-Booster (ChromoTek), FITC anti-mouse CD317 (PDCA-1; JF05-1C2.4.1) (Miltenyl Biotec), PE anti-mouse CD11c (HL3) (BD Biosciences), PE-Streptavidin and Cy5 anti-mouse B220 (TIB146; both homemade).

Techniques: Knock-Out, Transduction, Control, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

doi: 10.3389/fimmu.2018.01949

Figure Lengend Snippet: GCaMP6S functions as an efficient and specific sensor for Ca 2+ flux in Cas9 Hoxb8 cell-derived DCs. Ccr7 +/+ and Ccr7 −/− Cas9-Hoxb8 cells were transduced with a retrovirus expressing dTom and the Ca 2+ sensor GCaMP6S. Successfully transduced cells were sorted based on the expression of dTom and subsequently differentiated to mature DCs in the presence of GM-CSF followed by LPS treatment. GCaMP6S intensity was measured by flow cytometry. After recording a baseline for 45 s, cells were treated with 500 ng/ml CCL21 and signals were recorded for additional 240 s. Finally, 1 μg/ml of ionomycin was added and signals were acquired for another 60 s. Data are representative for three independent experiments.

Article Snippet: Following antibodies and staining reagents were used in this study: Brilliant Violet 510 anti-mouse I-A/I-E (clone M5/114.15.2), PE-Cy7 anti-mouse CD11c (N418), APC rat IgG2c κ Isotype control (RTK4174), PerCP-Streptavidin, PerCP-Cy5.5 anti-mouse CD8α (53–6.7), PerCP anti-mouse CD4 (RM4-5), APC anti-mouse CD40 (3/23), APC anti-mouse CD80 (16-10A1), APC Armenian Hamster IgG Isotype control (HTK888), FITC anti-mouse MHCII/I-Ab (AF6-120.1), APC rat IgG2b κ Isotype control (RTK4530), PE-Cy7 anti-mouse CD11b (N418) (all from Biolegend), PE anti-mouse CD11b (M1/70), PE anti-mouse TCR Vα2 (B20.1) (all from Invitrogen), eF660 anti-mouse CD11b (M1/70), eF450 anti-mouse CD11b (M1/70), PE anti-mouse F4/80 (BM8), PE anti-rat IgG2a k-Isotype control (BR2a), APC anti-mouse CXCR4 (2B11), Alexa Fluor 488 anti-mouse Lyve-1 (ALY7), APC anti-mouse CD117/cKit (ACKα), PE anti-mouse CD135/Flt3 (A2F10), APC anti-mouse CD11c (N418), PE-Cy7 anti-mouse Ly-6A/E/Sca-1 (D7), biotin anti-mouse CD115/M-CSFR (AF598), APC anti-mouse Ly6C (HK1.4), PE-Cy7 anti-mouse CD45.1 (A20), biotin rat IgG2a κ Isotype control (eBR2a), PE rat IgG2b κ Isotype control (eB149/10H5), APC anti-mouse CD86 (GL1), APC rat IgG2a κ Isotype control (eBR2a), PE anti-mouse CD197 (CCR7) (4B12), APC anti-mouse CD197 (CCR7) (4B12), APC-eF780 anti-mouse Ly-6G/GR-1 (RB6-8C5; all from eBioscience), ATT0647 GFP-Booster (ChromoTek), FITC anti-mouse CD317 (PDCA-1; JF05-1C2.4.1) (Miltenyl Biotec), PE anti-mouse CD11c (HL3) (BD Biosciences), PE-Streptavidin and Cy5 anti-mouse B220 (TIB146; both homemade).

Techniques: Derivative Assay, Transduction, Expressing, Flow Cytometry

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: High-parameter immune profiling with CyTOF

doi: 10.1007/978-1-4939-9773-2_16

Figure Lengend Snippet: Reference Panel (Hartmann et al; Submitted)

Article Snippet: 167 , Er , CCR7 (CD197) , T Cell subset (eff/mem) , surface , Fluidigm , 100 , 3167009A.

Techniques: Marker, Staining, Clinical Proteomics

Journal: Scientific Reports

Article Title: Salmonella enterica serovar Typhi exposure elicits ex vivo cell-type-specific epigenetic changes in human gut cells

doi: 10.1038/s41598-020-70492-2

Figure Lengend Snippet: Unsupervised clustering of epigenetic changes between and within the different mucosal cell subsets after wild-type S . Typhi infections. Cells isolated cells from healthy terminal ileum surgical tissues were exposed to S . Typhi strain Ty2 (Ty2) at 1:100 MOI. Cells cultured with media only were used as controls (media). After 3 h of incubation, cells were stained using a panel of 33 metal-labeled Abs, and the chromatin modifications were analyzed at the single-cell level by mass cytometry. To visualize the clustering of the 11 cell subsets ( i.e ., B-, CD3+ T-, CD4+ T-, CD8 + T-, NK, TCR-γδ, Mucosal associated invariant (MAIT) and NKT cells, as well as monocytes, macrophages, and epithelial cells), tSNE-defined population distributions and clustering were colored by meta-cluster. ( A ) t-SNE maps of chromatin modifications. Settings to run the t-SNE algorithm were set-up in Cytobank. ( B ) Color-coded key showing the location of epigenetic marks meta-clusters. Data are representative of one out of three experiments with terminal ileum segments from 2 different donors, one replicate each.

Article Snippet: Cells were surface stained with anti-human mAbs to CD3 (clone UCHT1), CD4 (clone SK3), CD8 (clone RPA-T8), CD11b (clone ICRF44), CD16 (clone 3G8), CD19 (clone HIB19), CD38 (clone HIT2), CD45 (clone HI30), CD45RO (clone UCHL), CD56 (clone HCD56), CD57 (clone HCD57), CD69 (clone FN50), CD161 (clone HP-3G10), CD163 (clone GHI/61), CCR7 (clone G043H7), EpCAM (CD326, clone 9C4), HLA-DR (clone L243), TCR γδ (clone 11F2)(Fluidigm, Sunnyvale, CA), CD14 (clone 3C10) (Invitrogen, Carlsbad, CA), and TCR Vα7.2 (clone 3C10) (Biolegend, San Diego, CA).

Techniques: Isolation, Cell Culture, Incubation, Staining, Labeling, Mass Cytometry

Journal: Scientific Reports

Article Title: Salmonella enterica serovar Typhi exposure elicits ex vivo cell-type-specific epigenetic changes in human gut cells

doi: 10.1038/s41598-020-70492-2

Figure Lengend Snippet: Chromatin profiles of the epigenetic changes in TCR-γδ cells induced by S . Typhi. Cells isolated cells from healthy terminal ileum surgical tissues were exposed to S . Typhi strain Ty2 (Ty2) and cultured as described in Fig. . Cells cultured with media only were used as controls (media). FCOM data of the 28 combinations within the acceptability criteria for changes of the chromatin marks are shown. Bars represent the net difference ( S . Typhi-infected minus uninfected cultures). Bar graphs extend from the 25th to 75th percentiles; the line in the middle represents the median of the pooled data. The whiskers delineate the smallest to the largest value. Data are representative of three experiments with terminal ileum segments from 4 different donors, one replicate each. P values < 0.05 were considered significant (red-colored boxes).

Article Snippet: Cells were surface stained with anti-human mAbs to CD3 (clone UCHT1), CD4 (clone SK3), CD8 (clone RPA-T8), CD11b (clone ICRF44), CD16 (clone 3G8), CD19 (clone HIB19), CD38 (clone HIT2), CD45 (clone HI30), CD45RO (clone UCHL), CD56 (clone HCD56), CD57 (clone HCD57), CD69 (clone FN50), CD161 (clone HP-3G10), CD163 (clone GHI/61), CCR7 (clone G043H7), EpCAM (CD326, clone 9C4), HLA-DR (clone L243), TCR γδ (clone 11F2)(Fluidigm, Sunnyvale, CA), CD14 (clone 3C10) (Invitrogen, Carlsbad, CA), and TCR Vα7.2 (clone 3C10) (Biolegend, San Diego, CA).

Techniques: Isolation, Cell Culture, Infection

Journal: Cell metabolism

Article Title: Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue

doi: 10.1016/j.cmet.2022.02.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD197 , Fluidigm , Cat# 3164013a.

Techniques: Immunofluorescence, Recombinant, Cytometry, Purification, Virus, Control, shRNA, Liposomes, Cell Culture, Red Blood Cell Lysis, XF Assay, Staining, Isolation, Transfection, Live Cell Imaging, Mouse Assay, Software, Gene Expression

Journal: PLoS ONE

Article Title: CCL21/CCR7 Enhances the Proliferation, Migration, and Invasion of Human Bladder Cancer T24 Cells

doi: 10.1371/journal.pone.0119506

Figure Lengend Snippet: Effect of CCL21 on T24 cell proliferation.

Article Snippet: CCL21 recombinant human protein was purchased from Perprotech (Rocky Hill, NJ, USA) and polyclonal rabbit anti-human CCR7 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Control

Journal: PLoS ONE

Article Title: CCL21/CCR7 Enhances the Proliferation, Migration, and Invasion of Human Bladder Cancer T24 Cells

doi: 10.1371/journal.pone.0119506

Figure Lengend Snippet: The assay was performed using a transwell chamber coated with 30 μl of Matrigel mixture solution with five different groups consisting of vehicle control, CCR7 antibody 20 ng/ml plus CCL21 50 ng/ml, CCL21 50 ng/ml, CCL21 100 ng/ml, and CCL21 200 ng/ml. The data present Mean ± SD from four independent experiments.

Article Snippet: CCL21 recombinant human protein was purchased from Perprotech (Rocky Hill, NJ, USA) and polyclonal rabbit anti-human CCR7 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Control

Journal: PLoS ONE

Article Title: CCL21/CCR7 Enhances the Proliferation, Migration, and Invasion of Human Bladder Cancer T24 Cells

doi: 10.1371/journal.pone.0119506

Figure Lengend Snippet: Lane 1: T24 cells treated with vehicle control; Lane 2: T24 cells treated with CCR7 antibody 20 ng/ml plus CCL21 50 ng/ml; Lane 3: T24 cells treated with 50 ng/ml CCL21; Lane 4: T24 cells treated with 100 ng/ml CCL21; and Lane 5: T24 cells treated with 200 ng/ml CCL21. Cell lysates were prepared and run on 5–15% SDS-PAGE gels following by a Western blot analysis. Beta-actin was used as a loading control. The blots shown are representative of three independent experiments.

Article Snippet: CCL21 recombinant human protein was purchased from Perprotech (Rocky Hill, NJ, USA) and polyclonal rabbit anti-human CCR7 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Control, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: CCL21/CCR7 Enhances the Proliferation, Migration, and Invasion of Human Bladder Cancer T24 Cells

doi: 10.1371/journal.pone.0119506

Figure Lengend Snippet: Effects of CCL21/CCR7 on MMP-2 and MMP-9 expression as represented via OD values.

Article Snippet: CCL21 recombinant human protein was purchased from Perprotech (Rocky Hill, NJ, USA) and polyclonal rabbit anti-human CCR7 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: CCL21/CCR7 Enhances the Proliferation, Migration, and Invasion of Human Bladder Cancer T24 Cells

doi: 10.1371/journal.pone.0119506

Figure Lengend Snippet: T24 cells were treated with vehicle control, CCR7 antibody 20 ng/ml plus CCL21 50 ng/ml, CCL21 50 ng/ml, CCL21 100 ng/ml, and CCL21 200 ng/ml. The values (OD) present Mean ± SD from three independent experiments.

Article Snippet: CCL21 recombinant human protein was purchased from Perprotech (Rocky Hill, NJ, USA) and polyclonal rabbit anti-human CCR7 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Control

Journal: PLoS ONE

Article Title: CCL21/CCR7 Enhances the Proliferation, Migration, and Invasion of Human Bladder Cancer T24 Cells

doi: 10.1371/journal.pone.0119506

Figure Lengend Snippet: Antiapoptotic effects of CCL21/CCR7 on T24.

Article Snippet: CCL21 recombinant human protein was purchased from Perprotech (Rocky Hill, NJ, USA) and polyclonal rabbit anti-human CCR7 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

doi: 10.3389/fimmu.2018.01949

Figure Lengend Snippet: CCR7-induced Ca 2+ signaling during entry of Cas9-Hoxb8-DCs transduced with Ca 2+ sensor GCaMP6S into the lymph node. (A) Ex vivo time-lapse imaging of lymph nodes within 5 min after intralymphatic injection of 4 × 10 4 Ccr7 +/+ Hoxb8-DCs (left) or Ccr7 −/− Hoxb8-DCs (right) transduced with Ca 2+ sensor GCaMP6S. White arrowheads indicate cells with changes in GCaMP6S signal intensity, indicating changes in Ca 2+ concentration within the cell. For more details see Supplementary Video . Scale bar represents 15 μm. (B) Changes in GCaMP6S signal intensity for each 5 GCaMP6S + Ccr7 +/+ Cas9-Hoxb8-DCs and GCaMP6S + Ccr7 −/− Cas9-Hoxb8-DCs. Data are represented as a difference in GCaMP6S signal intensity for each time point to the median value for next 3 min. Horizontal dashed lines depict thresholds [defined as a change in a signal intensity >1,000 arbitrary units (a.u.)] used to detect Ca 2+ signals. (C) Pie-charts indicating the percentage of GCaMP6S + Ccr7 +/+ Cas9-Hoxb8-DCs or GCaMP6S + Ccr7 −/− Cas9-Hoxb8-DCs with changes in Ca 2+ signals. There is a significant difference between groups ( p < 0.001, two-tailed Fisher's exact test). (D) Number and (E) average duration of Ca 2+ signals (changes in GCaMP6S intensity) per cell for the tracks with at least one recorded Ca 2+ signal. In (D,E) dots represent individual cells and red line median group value. Asterisk (*) indicates significant difference ( p < 0.05), while ns indicates no significant difference (Mann–Whitney test). Data are representative (A,B) or pool (C–E) of 6 independent experiments with total of 7 lymph nodes per cell type.

Article Snippet: Following antibodies and staining reagents were used in this study: Brilliant Violet 510 anti-mouse I-A/I-E (clone M5/114.15.2), PE-Cy7 anti-mouse CD11c (N418), APC rat IgG2c κ Isotype control (RTK4174), PerCP-Streptavidin, PerCP-Cy5.5 anti-mouse CD8α (53–6.7), PerCP anti-mouse CD4 (RM4-5), APC anti-mouse CD40 (3/23), APC anti-mouse CD80 (16-10A1), APC Armenian Hamster IgG Isotype control (HTK888), FITC anti-mouse MHCII/I-Ab (AF6-120.1), APC rat IgG2b κ Isotype control (RTK4530), PE-Cy7 anti-mouse CD11b (N418) (all from Biolegend), PE anti-mouse CD11b (M1/70), PE anti-mouse TCR Vα2 (B20.1) (all from Invitrogen), eF660 anti-mouse CD11b (M1/70), eF450 anti-mouse CD11b (M1/70), PE anti-mouse F4/80 (BM8), PE anti-rat IgG2a k-Isotype control (BR2a), APC anti-mouse CXCR4 (2B11), Alexa Fluor 488 anti-mouse Lyve-1 (ALY7), APC anti-mouse CD117/cKit (ACKα), PE anti-mouse CD135/Flt3 (A2F10), APC anti-mouse CD11c (N418), PE-Cy7 anti-mouse Ly-6A/E/Sca-1 (D7), biotin anti-mouse CD115/M-CSFR (AF598), APC anti-mouse Ly6C (HK1.4), PE-Cy7 anti-mouse CD45.1 (A20), biotin rat IgG2a κ Isotype control (eBR2a), PE rat IgG2b κ Isotype control (eB149/10H5), APC anti-mouse CD86 (GL1), APC rat IgG2a κ Isotype control (eBR2a), PE anti-mouse CD197 (CCR7) (4B12), APC anti-mouse CD197 (CCR7) (4B12), APC-eF780 anti-mouse Ly-6G/GR-1 (RB6-8C5; all from eBioscience), ATT0647 GFP-Booster (ChromoTek), FITC anti-mouse CD317 (PDCA-1; JF05-1C2.4.1) (Miltenyl Biotec), PE anti-mouse CD11c (HL3) (BD Biosciences), PE-Streptavidin and Cy5 anti-mouse B220 (TIB146; both homemade).

Techniques: Transduction, Ex Vivo, Imaging, Injection, Concentration Assay, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

doi: 10.3389/fimmu.2018.01949

Figure Lengend Snippet: Phenotypic and functional verification of CRISPR/Cas9-mediated knockout of Ccr7 in Hoxb8 cell-derived DCs. (A) Cas9-Hoxb8 cells were transduced with a dTom- and CCR7gRNA-encoding lentivirus. Successfully transduced cells were sorted based on the expression of dTom and subsequently differentiated to mature DCs in the presence of GM-CSF followed by treatment with LPS. Ccr7 +/+ and Ccr7 −/− DCs were analyzed by flow cytometry for their expression of CD80 and CCR7. Data are representative for four independent experiments. (B) Analysis of the composition and frequency of insertions and deletions of Ccr7 −/− Cas9-Hoxb8 cells. R, Pearson correlation coefficient; R 2 describes how strongly the calculated chromatograph of the indel distribution correlates with the Sanger sequencing results of the sample DNA. (C) Chemotactic migration of Ccr7 +/+ and Ccr7 −/− DCs for 2 h toward medium alone or 10, 100, and 200 ng/ml CCL21. Data are pooled from 3 independent experiments with n = 8 in total. Mean + SEM; Kruskal–Wallis and Dunn's multiple comparisons test; ns, not significant; ** p < 0.01. (D) Microscopy of popliteal lymph nodes obtained 4 h after intralymphatic injection of YFP-expressing Ccr7 +/+ DCs and dTom-expressing Ccr7 −/− DCs (5–8 × 10 4 cells in 5 μl PBS; scale bar: 200 μm). (E) Total cell counts and (F) relative distribution of Ccr7 +/+ and Ccr7 −/− DCs 4 h after intralymphatic injection into popliteal LNs of B6 mice. (G) Migration distance from the subcapsular sinus (SCS) for the Ccr7 +/+ and Ccr7 −/− DCs that entered LN parenchyma. Dots represent cell number per LN section (E) or individual cells (G) . Data are representative for (D) or pooled from (E–G) two independent experiments with a total of 7 lymph nodes analyzed. Error bars, SD; red bars, median; Mann–Whitney test; * p < 0.05; **** p < 0.0001.

Article Snippet: Following antibodies and staining reagents were used in this study: Brilliant Violet 510 anti-mouse I-A/I-E (clone M5/114.15.2), PE-Cy7 anti-mouse CD11c (N418), APC rat IgG2c κ Isotype control (RTK4174), PerCP-Streptavidin, PerCP-Cy5.5 anti-mouse CD8α (53–6.7), PerCP anti-mouse CD4 (RM4-5), APC anti-mouse CD40 (3/23), APC anti-mouse CD80 (16-10A1), APC Armenian Hamster IgG Isotype control (HTK888), FITC anti-mouse MHCII/I-Ab (AF6-120.1), APC rat IgG2b κ Isotype control (RTK4530), PE-Cy7 anti-mouse CD11b (N418) (all from Biolegend), PE anti-mouse CD11b (M1/70), PE anti-mouse TCR Vα2 (B20.1) (all from Invitrogen), eF660 anti-mouse CD11b (M1/70), eF450 anti-mouse CD11b (M1/70), PE anti-mouse F4/80 (BM8), PE anti-rat IgG2a k-Isotype control (BR2a), APC anti-mouse CXCR4 (2B11), Alexa Fluor 488 anti-mouse Lyve-1 (ALY7), APC anti-mouse CD117/cKit (ACKα), PE anti-mouse CD135/Flt3 (A2F10), APC anti-mouse CD11c (N418), PE-Cy7 anti-mouse Ly-6A/E/Sca-1 (D7), biotin anti-mouse CD115/M-CSFR (AF598), APC anti-mouse Ly6C (HK1.4), PE-Cy7 anti-mouse CD45.1 (A20), biotin rat IgG2a κ Isotype control (eBR2a), PE rat IgG2b κ Isotype control (eB149/10H5), APC anti-mouse CD86 (GL1), APC rat IgG2a κ Isotype control (eBR2a), PE anti-mouse CD197 (CCR7) (4B12), APC anti-mouse CD197 (CCR7) (4B12), APC-eF780 anti-mouse Ly-6G/GR-1 (RB6-8C5; all from eBioscience), ATT0647 GFP-Booster (ChromoTek), FITC anti-mouse CD317 (PDCA-1; JF05-1C2.4.1) (Miltenyl Biotec), PE anti-mouse CD11c (HL3) (BD Biosciences), PE-Streptavidin and Cy5 anti-mouse B220 (TIB146; both homemade).

Techniques: Functional Assay, CRISPR, Knock-Out, Derivative Assay, Transduction, Expressing, Flow Cytometry, Sequencing, Migration, Microscopy, Injection, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

doi: 10.3389/fimmu.2018.01949

Figure Lengend Snippet: The unlimited proliferative capacity of Cas9-Hoxb8 cells allows the consecutive knockout of multiple genes. Ccr7 −/− Cas9-Hoxb8 cells were transduced with a Cerulean- and CXCR4-gRNA-encoding lentivirus. Transduced cells as well as control Cas9-Hoxb8 cells were subsequently differentiated to mature DCs in the presence of GM-CSF followed by the treatment with LPS. DCs were analyzed by flow cytometry for their expression of CD80, CCR7, and CXCR4. Gray curves depict isotype controls. Data are representative of two independent experiments.

Article Snippet: Following antibodies and staining reagents were used in this study: Brilliant Violet 510 anti-mouse I-A/I-E (clone M5/114.15.2), PE-Cy7 anti-mouse CD11c (N418), APC rat IgG2c κ Isotype control (RTK4174), PerCP-Streptavidin, PerCP-Cy5.5 anti-mouse CD8α (53–6.7), PerCP anti-mouse CD4 (RM4-5), APC anti-mouse CD40 (3/23), APC anti-mouse CD80 (16-10A1), APC Armenian Hamster IgG Isotype control (HTK888), FITC anti-mouse MHCII/I-Ab (AF6-120.1), APC rat IgG2b κ Isotype control (RTK4530), PE-Cy7 anti-mouse CD11b (N418) (all from Biolegend), PE anti-mouse CD11b (M1/70), PE anti-mouse TCR Vα2 (B20.1) (all from Invitrogen), eF660 anti-mouse CD11b (M1/70), eF450 anti-mouse CD11b (M1/70), PE anti-mouse F4/80 (BM8), PE anti-rat IgG2a k-Isotype control (BR2a), APC anti-mouse CXCR4 (2B11), Alexa Fluor 488 anti-mouse Lyve-1 (ALY7), APC anti-mouse CD117/cKit (ACKα), PE anti-mouse CD135/Flt3 (A2F10), APC anti-mouse CD11c (N418), PE-Cy7 anti-mouse Ly-6A/E/Sca-1 (D7), biotin anti-mouse CD115/M-CSFR (AF598), APC anti-mouse Ly6C (HK1.4), PE-Cy7 anti-mouse CD45.1 (A20), biotin rat IgG2a κ Isotype control (eBR2a), PE rat IgG2b κ Isotype control (eB149/10H5), APC anti-mouse CD86 (GL1), APC rat IgG2a κ Isotype control (eBR2a), PE anti-mouse CD197 (CCR7) (4B12), APC anti-mouse CD197 (CCR7) (4B12), APC-eF780 anti-mouse Ly-6G/GR-1 (RB6-8C5; all from eBioscience), ATT0647 GFP-Booster (ChromoTek), FITC anti-mouse CD317 (PDCA-1; JF05-1C2.4.1) (Miltenyl Biotec), PE anti-mouse CD11c (HL3) (BD Biosciences), PE-Streptavidin and Cy5 anti-mouse B220 (TIB146; both homemade).

Techniques: Knock-Out, Transduction, Control, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

doi: 10.3389/fimmu.2018.01949

Figure Lengend Snippet: GCaMP6S functions as an efficient and specific sensor for Ca 2+ flux in Cas9 Hoxb8 cell-derived DCs. Ccr7 +/+ and Ccr7 −/− Cas9-Hoxb8 cells were transduced with a retrovirus expressing dTom and the Ca 2+ sensor GCaMP6S. Successfully transduced cells were sorted based on the expression of dTom and subsequently differentiated to mature DCs in the presence of GM-CSF followed by LPS treatment. GCaMP6S intensity was measured by flow cytometry. After recording a baseline for 45 s, cells were treated with 500 ng/ml CCL21 and signals were recorded for additional 240 s. Finally, 1 μg/ml of ionomycin was added and signals were acquired for another 60 s. Data are representative for three independent experiments.

Article Snippet: Following antibodies and staining reagents were used in this study: Brilliant Violet 510 anti-mouse I-A/I-E (clone M5/114.15.2), PE-Cy7 anti-mouse CD11c (N418), APC rat IgG2c κ Isotype control (RTK4174), PerCP-Streptavidin, PerCP-Cy5.5 anti-mouse CD8α (53–6.7), PerCP anti-mouse CD4 (RM4-5), APC anti-mouse CD40 (3/23), APC anti-mouse CD80 (16-10A1), APC Armenian Hamster IgG Isotype control (HTK888), FITC anti-mouse MHCII/I-Ab (AF6-120.1), APC rat IgG2b κ Isotype control (RTK4530), PE-Cy7 anti-mouse CD11b (N418) (all from Biolegend), PE anti-mouse CD11b (M1/70), PE anti-mouse TCR Vα2 (B20.1) (all from Invitrogen), eF660 anti-mouse CD11b (M1/70), eF450 anti-mouse CD11b (M1/70), PE anti-mouse F4/80 (BM8), PE anti-rat IgG2a k-Isotype control (BR2a), APC anti-mouse CXCR4 (2B11), Alexa Fluor 488 anti-mouse Lyve-1 (ALY7), APC anti-mouse CD117/cKit (ACKα), PE anti-mouse CD135/Flt3 (A2F10), APC anti-mouse CD11c (N418), PE-Cy7 anti-mouse Ly-6A/E/Sca-1 (D7), biotin anti-mouse CD115/M-CSFR (AF598), APC anti-mouse Ly6C (HK1.4), PE-Cy7 anti-mouse CD45.1 (A20), biotin rat IgG2a κ Isotype control (eBR2a), PE rat IgG2b κ Isotype control (eB149/10H5), APC anti-mouse CD86 (GL1), APC rat IgG2a κ Isotype control (eBR2a), PE anti-mouse CD197 (CCR7) (4B12), APC anti-mouse CD197 (CCR7) (4B12), APC-eF780 anti-mouse Ly-6G/GR-1 (RB6-8C5; all from eBioscience), ATT0647 GFP-Booster (ChromoTek), FITC anti-mouse CD317 (PDCA-1; JF05-1C2.4.1) (Miltenyl Biotec), PE anti-mouse CD11c (HL3) (BD Biosciences), PE-Streptavidin and Cy5 anti-mouse B220 (TIB146; both homemade).

Techniques: Derivative Assay, Transduction, Expressing, Flow Cytometry