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: CNS Neuroscience & Therapeutics

Article Title: Atractylenolide III ameliorates spinal cord injury in rats by modulating microglial/macrophage polarization

doi: 10.1111/cns.13839

Figure Lengend Snippet: Table of antibodies used

Article Snippet: CCR7 , Rabbit polyclonal , bs−1305R , AB_11056896 , PE , Bioss , 1 μg/test.

Techniques: Conjugation Assay, Concentration Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Atractylenolide III ameliorates spinal cord injury in rats by modulating microglial/macrophage polarization

doi: 10.1111/cns.13839

Figure Lengend Snippet: ATL‐III regulated the M1/M2 polarization of microglia/macrophages in rats after SCI. (A–H) Typical immunohistofluorescence pictures of CD68 (green), CCR7 (red in A–C), Arg‐1 (red in E–G), and nuclei (blue) in the spinal cords of sham, control, and ATL‐III rats. (D,H) The numbers of CD68 + CCR7 + (M1 cells) and CD68 + Arg‐1 + (M2 cells) cells. (I–K, M–O) Typical flow cytometry images of myeloid tissues from sham, control, and ATL‐III rats. (L,P) Proportions of CD68 + CCR7 + and CD68 + CD206 + cells. The data are presented as the mean ± SD ( n = 6). * p < 0.05, ** p < 0.01 compared with the control rats

Article Snippet: CCR7 , Rabbit polyclonal , bs−1305R , AB_11056896 , PE , Bioss , 1 μg/test.

Techniques: Immunohistofluorescence, Flow Cytometry

Journal: Discover Oncology

Article Title: Integrated WGCNA retrieval of T-cell exhaustion genes related to radiosensitivity in locally advanced cervical cancer and prediction of immunotherapy efficacy

doi: 10.1007/s12672-025-03673-y

Figure Lengend Snippet: IHC assay results of the protein expression levels of APIBEC3H, CCR7, PILRA, and SLC15A3 in LACC patients

Article Snippet: Subsequently, the sections were incubated at 4 °C overnight with the corresponding primary antibody against APOBEC3H (1:200, PA5-54426, Invitrogen, USA), CCR7 (1:100, 25898-1-AP, Proteintech, China), PLRA (1:100, PA5-147474, Invitrogen, USA), SLC15A3 (1:100, PA5-61614, Invitrogen, USA).

Techniques: Expressing

Journal: Cell

Article Title: Single-cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment

doi: 10.1016/j.cell.2018.05.060

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-CD197/CCR7 167Er , Fluidigm , Cat:3167009A.

Techniques: Staining, Antibody Labeling, Software, Diffusion-based Assay

Journal: Nature metabolism

Article Title: The glucose transporter 2 regulates CD8 + T cell function via environment sensing.

doi: 10.1038/s42255-023-00913-9

Figure Lengend Snippet: Fig. 8 | Glut2 is expressed by, and is functional in, human T cells. a–d, GLUT2 expression (a and b) and 6-NBDG uptake (c and d) by human CD4+ and CD8+ T cells from WT and age-matched and sex-matched homozygous carriers of SLC2A2 SNP (HO) ex vivo (a and c) or after 2 d of antibody activation in complete RPMI (R1) or glucose-free medium reconstituted with the indicated concentrations of glucose (b and d); representative histograms (grey line indicates controls, red line indicates SLC2A2 SNP carriers, black indicates FMO control staining) and mean data measured in the indicated number of individuals ± s.e.m., n = 4–10 per group, N = 1. e–h, Representative dot plots and percentage of circulating human CD4+ and CD8+ T cells (e) and their subsets (f and g, respectively) in WT and HO individuals: regulatory T cells (CD4+CD25hiCD127lo), naive (CD45RA+CCR7+), central memory (TCM, CD45RA−CCR7+), effector memory (TEM, CD45RA−CCR7−) and terminally differentiated effector memory T cells (TEMRA, CD45RA+CCR7−; ±s.d.; n = 7 per group, N = 1). i, CFSE (5 µM)-labelled human CD8+ and CD4+ T cells were activated by antibodies for 4 d in the presence of Glut1 inhibitor (STF-31, 1.25 µM) or dual inhibitor (phloretin, 75 µM) or vehicle added for the last 24 h. Representative histograms and mean data representative of three independent

Article Snippet: NB110-39113AF405); Glut2 PE (clone 205115, NOVUS, FAB1440P); Gal-9 APC (clone 108A2, BioLegend, 137912) or PE/Cy7 (clone RG9-35, eBioscience, 25-9211-80); Stomatin AF488 (polyclonal, Bioss ANTIBODIES, bs-10443R-A488); MHCI (H-2kb/H-2Db) AF647 (clone 28-8- BV605 (clone H57-597, BioLegend, 109241 ); CCR4 APC (clone 2G12, BioLegend, 131211); CCR5 PE (clone HM-CCR5, eBioscience, 12-1951-81); CCR6 PE (clone 29-2L17, BioLegend, 129804); CCR7 APC (clone 4B12, BioLegend, 120107); CXCR3 PerCP/Cy5.5 (clone CXCR3-173, BioLegend, 126514); CXCR4 FITC (clone 2B11/CXCR4, BD biosciences, 551967); LFA-1 PE/Cy7 (clone M17/4, BioLegend, 101122); B220 BV605 (clone RA3- 1D4B, BioLegend, 121624); Phenotypic characterization of human cells was performed with: CD3 AF700 (clone UCHT1, Invitrogen, 56-0038-80); CD4 BV605 (clone SK3, also known as Leu3a, BD Biosciences, 565998); CD8 ef450 (clone SK1, BD Biosciences, 48 -0087-42); CD8 FITC (clone RPA-T8, BioLegend, 301060); CCR7 FITC (clone REA546, Miltenyi Biotec, 130-120-468); CD45RA-BV785 (clone HI100); CD45RA-PerCPVio700 (clone REA1047, BioLegend, 304140); CD45RO- APCVio770 (clone REA611, Miltenyi biotec, 130-113- 547); Glut2-PE (clone 199017, NOVUS, FAB1414P) Intracellular staining for murine studies was performed with the following antibodies: FoxP3 APC (clone FJK-16s, eBioscience, 77- 5775- GB1 -17 ef450 (clone eBio17B7, eBioscience, 48-7177- 557729) was used for intracellular cytokine staining in human cells.

Techniques: Functional Assay, Expressing, Ex Vivo, Activation Assay, Control, Staining

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: 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: 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: Frontiers in Bioengineering and Biotechnology

Article Title: Weakly acidic microenvironment of the wound bed boosting the efficacy of acidic fibroblast growth factor to promote skin regeneration

doi: 10.3389/fbioe.2023.1150819

Figure Lengend Snippet: The application of aFGF- or bFGF- loaded GelMA hydrogels with different pH values on wound closure. (A) The maintenance of weak acid, neutral, and alkaline microenvironments of wound sites filled with GelMA hydrogels with different pH values. (B) The photographs and H&E staining of the healed wound treated with aFGF- or bFGF- loaded GelMA hydrogels with different pH values for 8 days. (C) The wound closure rate of aFGF- or bFGF- loaded GelMA hydrogels with different pH values after 4 and 8 days of treatment. (D) The H&E staining results of aFGF- or bFGF- loaded GelMA hydrogels with different pH values after 4 and 8 days of treatment. (E) The re-epithelialization rate of aFGF- or bFGF- loaded GelMA hydrogels with different pH values after 4 and 8 days of treatment. (F,G) The expression of α-SMA within the wound area of aFGF- or bFGF- loaded GelMA hydrogels with different pH values after 4 and 8 days of treatment. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies included α-SMA (Boster, China, Catalog#BM0002), CD31(Proteintech, China, Catalog#28083-1-AP), CCR7 (Abcam, United States ; Catalog#EPR23192-57), and CD206 (Abcam, United States ; Catalog#EPR25215-277).

Techniques: Staining, Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Weakly acidic microenvironment of the wound bed boosting the efficacy of acidic fibroblast growth factor to promote skin regeneration

doi: 10.3389/fbioe.2023.1150819

Figure Lengend Snippet: The granulation tissue formation of the wounds treated with aFGF- or bFGF- loaded GelMA hydrogels with different pH values for 4 and 8 days. (A) The trichrome staining of the wounds treated with aFGF- or bFGF- loaded GelMA hydrogels with different pH values for 4, 8, and 12 days. (B) The immunohistochemical staining of α-SMA within the wounds treated with aFGF- or bFGF- loaded GelMA hydrogels with different pH values for 4 and 8 days. (C) The quantification of the α-SMA expression of the (B) . * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies included α-SMA (Boster, China, Catalog#BM0002), CD31(Proteintech, China, Catalog#28083-1-AP), CCR7 (Abcam, United States ; Catalog#EPR23192-57), and CD206 (Abcam, United States ; Catalog#EPR25215-277).

Techniques: Staining, Immunohistochemical staining, Expressing