Journal: Frontiers in Cellular Neuroscience

Article Title: The Time Course of Monocytes Infiltration After Acoustic Overstimulation

doi: 10.3389/fncel.2022.844480

Figure Lengend Snippet: Inflammatory monocytes from the bloodstream infiltrated the cochlea after acoustic overstimulation. (A) Effects of acoustic overstimulation (120 dB, 1 h white band noise exposure) on the permanent threshold shift ( N = 8 ears). (B) Effects of noise exposure on outer hair cell degeneration ( N = 4 ears in each group). (C) Effects of acoustic stimulation on peripheral blood CD11b + Ly6G – F4/80 + CX3CR1 + populations and distinction between Ly6C ++ inflammatory monocytes and Ly6C – patrolling monocytes. (D) Effects of acoustic overstimulation on cell populations (CD11b + Ly6G – F4/80 + CX3CR1 + cells; Ly6C ++ possible inflammatory monocytes and Ly6C – possible resident macrophages) in cochlea at 2 days. (E) Clodronate liposome successfully reduced monocytes in peripheral blood (left) compared to neutrophils (right) in 1 dpn at which the monocytes begin to infiltrate to the cochlea. (F) Effects of clodronate liposome treatment after acoustic overstimulation on Ly6C ++ possible inflammatory monocyte populations in the cochlea after 2 days. (left) Effects of clodronate liposome treatment after acoustic overstimulation on Ly6C – possible resident macrophage populations. (right) Clod : clodronate liposome-treated group. ** : p < 0.01, *: p < 0.05, ns : not significant. (G) Immunofluorescence study comparing control and clodronate liposome at 2 dpn. White arrows indicate clusters of monocytes. The black box in the light microscopy image located on the left indicates the anatomical location of the immunofluorescence images. Scale bar = 50 μm.

Article Snippet: 7-Aminoactinomycin D (7-AAD; cat. no. 420403; Biolegend) was used for live cell gating, phycoerythrin (PE)-Cy7-conjugated anti-CD11b (M1/70; cat. no. 25-0112-82; Invitrogen, Carlsbad, CA, United States) was used for myeloid cell gating, PE-conjugated anti-Ly6G (1A8; cat. no. 127608; Biolegend) was used for neutrophil gating, APC-conjugated anti-F4/80 (BM8; cat. no. 17-4801-82; Invitrogen) was used for monocyte/macrophage gating, and APC-Cy7-conjugated anti-Ly6C (HK1.4; cat. no. 128026; Biolegend) was used for inflammatory monocyte gating ( , ).

Techniques: Immunofluorescence, Light Microscopy

Journal: Frontiers in Cellular Neuroscience

Article Title: The Time Course of Monocytes Infiltration After Acoustic Overstimulation

doi: 10.3389/fncel.2022.844480

Figure Lengend Snippet: Inflammatory monocytes infiltrated within 2 days after acoustic stimulation, whereas neutrophil infiltration was not observed. (A) Percentages of cells sorted by Ly6C expression from the CD11b + Ly6G – F4/80 + CX3CR1 + population, demonstrating Ly6C ++ cell infiltration within 2 days after acoustic overstimulation. (B) Total cell counts showing the CD11b + Ly6G – F4/80 + CX3CR1 + population did not change after 2 dpn. (C) Distinction between Ly6G + CX3CR1 – neutrophils and CD11b + cells in the peripheral blood. (D) Lack of distinct neutrophils in the cochlea at 1 day after acoustic overstimulation compared with CD11b + cells. (E) Changes in the percentages and the cell count of neutrophils in cochlea after acoustic overstimulation. There was no statistically significant value. Ctr : control, N1d : 1 day after noise, N2d : 2 days after noise, N3d : 3 days after noise, N5d : 5 days after noise, Mo : monocytes, M φ: macrophages. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, when compared with the control. (F) Immunofluorescence study of lower portion of spiral ligament at 1 dpn. Neutrophil (white) is mixed in the cluster of inflammatory monocytes (red and weak green). Scale bar = 20 μm.

Article Snippet: 7-Aminoactinomycin D (7-AAD; cat. no. 420403; Biolegend) was used for live cell gating, phycoerythrin (PE)-Cy7-conjugated anti-CD11b (M1/70; cat. no. 25-0112-82; Invitrogen, Carlsbad, CA, United States) was used for myeloid cell gating, PE-conjugated anti-Ly6G (1A8; cat. no. 127608; Biolegend) was used for neutrophil gating, APC-conjugated anti-F4/80 (BM8; cat. no. 17-4801-82; Invitrogen) was used for monocyte/macrophage gating, and APC-Cy7-conjugated anti-Ly6C (HK1.4; cat. no. 128026; Biolegend) was used for inflammatory monocyte gating ( , ).

Techniques: Expressing, Cell Counting, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: The Time Course of Monocytes Infiltration After Acoustic Overstimulation

doi: 10.3389/fncel.2022.844480

Figure Lengend Snippet: Infiltrated monocytes transformed into macrophages within 5 days after acoustic stimulation. Concatenated contour plots (merging four biological replicates for each time point) showing CD11b + Ly6G – F4/80 + CX3CR1 + cells at different time points after acoustic overstimulation. Black contour : cochlea sample, red contour : control peripheral blood sample, dots : center of each population.

Article Snippet: 7-Aminoactinomycin D (7-AAD; cat. no. 420403; Biolegend) was used for live cell gating, phycoerythrin (PE)-Cy7-conjugated anti-CD11b (M1/70; cat. no. 25-0112-82; Invitrogen, Carlsbad, CA, United States) was used for myeloid cell gating, PE-conjugated anti-Ly6G (1A8; cat. no. 127608; Biolegend) was used for neutrophil gating, APC-conjugated anti-F4/80 (BM8; cat. no. 17-4801-82; Invitrogen) was used for monocyte/macrophage gating, and APC-Cy7-conjugated anti-Ly6C (HK1.4; cat. no. 128026; Biolegend) was used for inflammatory monocyte gating ( , ).

Techniques: Transformation Assay

Journal: iScience

Article Title: CX3CR1 deficiency-induced TIL tumor restriction as a novel addition for CAR-T design in solid malignancies

doi: 10.1016/j.isci.2023.106443

Figure Lengend Snippet: NKG2D-CAR-T co-expressing CX3CR1 has higher activation in vitro

Article Snippet: Mouse blood samples were collected weekly and stained with the following anti-human antibodies: EGFR AF488 (R&D systems catalog number FAB9577G), CX3CR1 APC (Thermo Fisher Scientific catalog number 17-6099-42, RRID: AB_11149136 ), CD45 BUV805 (BD Biosciences catalog number 612891, RRID: AB_2870179 ), Live/Dead Fixable Aqua, and NKG2D PE-Cy7 (BD Biosciences catalog number 562365, RRID: AB_11153309 ).

Techniques: Expressing, Activation Assay, In Vitro

Journal: iScience

Article Title: CX3CR1 deficiency-induced TIL tumor restriction as a novel addition for CAR-T design in solid malignancies

doi: 10.1016/j.isci.2023.106443

Figure Lengend Snippet: NKG2D-CAR-T co-expressing CX3CR1 reduces tumor burden in solid tumor murine models

Article Snippet: Mouse blood samples were collected weekly and stained with the following anti-human antibodies: EGFR AF488 (R&D systems catalog number FAB9577G), CX3CR1 APC (Thermo Fisher Scientific catalog number 17-6099-42, RRID: AB_11149136 ), CD45 BUV805 (BD Biosciences catalog number 612891, RRID: AB_2870179 ), Live/Dead Fixable Aqua, and NKG2D PE-Cy7 (BD Biosciences catalog number 562365, RRID: AB_11153309 ).

Techniques: Expressing

Journal: iScience

Article Title: CX3CR1 deficiency-induced TIL tumor restriction as a novel addition for CAR-T design in solid malignancies

doi: 10.1016/j.isci.2023.106443

Figure Lengend Snippet: Key resources table

Article Snippet: Mouse blood samples were collected weekly and stained with the following anti-human antibodies: EGFR AF488 (R&D systems catalog number FAB9577G), CX3CR1 APC (Thermo Fisher Scientific catalog number 17-6099-42, RRID: AB_11149136 ), CD45 BUV805 (BD Biosciences catalog number 612891, RRID: AB_2870179 ), Live/Dead Fixable Aqua, and NKG2D PE-Cy7 (BD Biosciences catalog number 562365, RRID: AB_11153309 ).

Techniques: Marker, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Isolation, Activation Assay, Staining, Over Expression, Construct, Plasmid Preparation, Software, Microscopy, Imaging, Flow Cytometry

Journal: Cells

Article Title: Chronic Pain Induced by Social Defeat Stress in Juvenile Mice Depends on TLR4

doi: 10.3390/cells14050350

Figure Lengend Snippet: SDS increases TLR4 dimers of spinal cord microglia and DRG macrophages. ( A ) TLR4 dimers (%) in SDS and no-SDS groups of microglia (CD11b + population) and ( B ) macrophages (CD11b + /F4-80 + population) on day 22 of the protocol. ( C ) Gating strategy for lumbar spinal cord cells, first live cells were gated (APC-Cy7 live/dead Ghost dye); second, microglia population (PercpCy 5.5-conjugated CD11b+), lastly—TLR4 monomers (PC7-conjugated TLR4/MD2) or TLR4 total (APC-conjugated total TLR4). ( D ) Gating strategy for dorsal root ganglia cells, first live cells were gated (APC-Cy7 live/dead Ghost dye); second, microglia population (PercpCy 5.5-conjugated CD11b+), third macrophages population (488-conjugated F4-80) and lastly—TLR4 monomers (PC7-conjugated TLR4/MD2) or TLR4 total (APC-conjugated total TLR4). Data represent mean ± SEM, t -test, SDS vs. no-SDS, * p < 0.05, ** p < 0.01, n = 6 (SDS), and n = 6 (no-SDS).

Article Snippet: Single-cell suspensions were incubated for 45 min on ice with the following antibodies: APC-Cy7 live/dead Ghost dye (#18452, Cell Signaling, Danvers, MA, USA) or 540 live/dead Ghost dye (#72086, Cell Signaling, Danvers, MA, USA); PC7-conjugated TLR4/MD2 (#MTS510; ThermoFisher, Waltham, MA, USA); APC-conjugated total TLR4 (#SA15-21; Biolegend, San Diego, CA, USA); 488-conjugated F4-80 (#123110; Biolegend, San Diego, CA, USA); and PercpCy 5.5-conjugated CD11b (#101228; Biolegend, San Diego, CA, USA).

Techniques:

Journal: Nutrients

Article Title: Annona muricata L.-Derived Polysaccharides as a Potential Adjuvant to a Dendritic Cell-Based Vaccine in a Thymoma-Bearing Model

doi: 10.3390/nu12061602

Figure Lengend Snippet: Immunization with ALP-treated DCs induces a robust anti-tumor effect and immunity. ( A ) Two weeks after the final immunization, single cells obtained from PBS-, iDC-, OVAs-DC-, and ALP/OVAs-DC-immunized mice were stimulated ex vivo with OVA peptides for 6 h. The percentage of OVA-specific CD3 + CD4 + and CD3 + CD8 + T cells producing CD107a, IFN-γ, IL-2, or TNF-α was analyzed in accord with the gating strategy described in . Pie charts indicate the mean frequencies of CD4 + and CD8 + T cells co-expressing IFN-γ, TNF-α, IL-2, and CD107a. ( B ) Serum samples were analyzed using ELISA for OVA 323–339 -specific IgG2a and IgG1 antibodies in each immunized mouse. The mean ± SD ( n = 6 mice/group) shown are representative of 2 independent experiments. ( C ) Tumor volumes in mice ( n = 10 mice/group) that received PBS (G1: white solid dot), iDC (G2: green solid dot), OVAs-DC (G3: red solid dot), and ALP/OVA-DCs (G4: blue solid dot) vs. E.G7. Tumor growth was identified by calculating the diameter of the tumor every 3 days for 30 days. The mean ± SD shown are representative of 2 independent experiments. SD; standard deviation.

Article Snippet: The cells were then stained using Live/Dead and with anti-CD3 (APC-Cy7, eBioscience), anti-CD4 (Alexa488), and anti-CD8 (PerCp-Cy5.5, eBioscience) mouse Abs (mAbs) for 30 min at 4 °C.

Techniques: Ex Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation