Journal: Scientific Reports

Article Title: Cell release during perfusion reflects cold ischemic injury in rat livers

doi: 10.1038/s41598-020-57589-4

Figure Lengend Snippet: Alterations in liver-resident immune cell release into the perfusate after cold ischemia. ( a ) Percentage of presumed Kupffer cells (left/orange), liver-resident natural killer cells (also known as pit cells) (middle/red), and dendritic cells (right/yellow) in the perfusate, relative to the total number of nucleated cells (TNCs) that are released into the perfusate from fresh (n = 4), 24-h-cold ischemic (CI) (n = 5), and 72-h-CI (n = 4) livers. The perfusate recirculated during 3 hours of subnormothermic machine perfusion. Stars denote statistical significance (two-way ANOVA, followed by Tukey’s post-hoc test): *0.01 < p < 0.05; **0.001 < p < 0.01; ***0.0001 < p < 0.001; ****p < 0.0001. Error bars: SEM. ( b ) Imaging flow cytometry for the quantification of presumed Kupffer cells (CD45+/CD105−/SE1−/CD14+) and pit cells (CD45+/NKRP1A+/CD3−/OX62−). Dendritic cells were manually selected from a CD45+/NKRP1A−/CD3−/OX62+ population (not shown). Left: fresh livers. Right: 24-h-CI livers. ( c ) Representative images of surface marker expression images of Kupffer cells (top), pit cells (middle), and dendritic cells (below). Scale bars: 5 µm.

Article Snippet: The aliquot for panel 3 was additionally stained for the detection of pit p cells and dendritic p cells with Alexa 405-conjugated NKRP1A (1:80; Novus Biologicals; Cat# NB100-65297AF405), FITC-conjugated CD3 (1:50; Invitrogen, Carlsbad, CA, USA; Cat# 11-0030-82), and PE-conjugated OX62 (1:100; Thermo Fisher; Cat# 12-1030-82) antibodies.

Techniques: Imaging, Flow Cytometry, Marker, Expressing

Journal: Scientific Reports

Article Title: The immune response after noise damage in the cochlea is characterized by a heterogeneous mix of adaptive and innate immune cells

doi: 10.1038/s41598-020-72181-6

Figure Lengend Snippet: Percentages of CD45 + immune cells in control and noise-exposed cochleae.

Article Snippet: The whole-mount preparations and sections were stained using the following antibodies at 1:50 to 1:100 dilution: goat anti-mouse CD45 (AF114, R&D Systems; a kind gift from Dr. Tejbeer Kaur), rat anti-mouse CD11b (MCA711, Bio-Rad), rat anti-mouse F4/80 (CI-A3-1: NB600-404SS, Novus Biologicals), rabbit monoclonal CD3e (SP7:SAB5500058, Sigma-Aldrich), rat anti-mouse CD45R/B220 (RA3- 6B2; 553083, BD Pharmingen), mouse monoclonal neutrophil elastase (NP57; sc-53388, Santa Cruz Biotechnology, Inc.), CD161/ NK1.1 Antibody (PK136; NB100-77528SS, Novus Biologicals), and rabbit polyclonal myosin VI (25-6791, Proteus BioSciences).

Techniques: Control

Journal: Scientific Reports

Article Title: The immune response after noise damage in the cochlea is characterized by a heterogeneous mix of adaptive and innate immune cells

doi: 10.1038/s41598-020-72181-6

Figure Lengend Snippet: Immune cell phenotypic markers.

Article Snippet: The whole-mount preparations and sections were stained using the following antibodies at 1:50 to 1:100 dilution: goat anti-mouse CD45 (AF114, R&D Systems; a kind gift from Dr. Tejbeer Kaur), rat anti-mouse CD11b (MCA711, Bio-Rad), rat anti-mouse F4/80 (CI-A3-1: NB600-404SS, Novus Biologicals), rabbit monoclonal CD3e (SP7:SAB5500058, Sigma-Aldrich), rat anti-mouse CD45R/B220 (RA3- 6B2; 553083, BD Pharmingen), mouse monoclonal neutrophil elastase (NP57; sc-53388, Santa Cruz Biotechnology, Inc.), CD161/ NK1.1 Antibody (PK136; NB100-77528SS, Novus Biologicals), and rabbit polyclonal myosin VI (25-6791, Proteus BioSciences).

Techniques:

Journal: The Journal of Immunology Author Choice

Article Title: LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation

doi: 10.4049/jimmunol.1502462

Figure Lengend Snippet: CD161 is expressed on FDCs and on a subset of tonsillar T cells. ( A ) CD161 expression negatively correlated with CXCR5 expression on CD45RA − T cells (CD3 + CD4+) assessed by FACS ( n = 5, three independent experiments) and real-time PCR ( n = 3, two independent experiments). ( B ) Costaining of CD161 and CD3, showing no double-positives within the GC. Original magnification ×20. ( C ) Immunofluorescence staining of frozen human tonsils with an FDC marker (clone CNA.42) and CD161 (clone IMG14F1F11) showed complete overlap between these two markers. Original magnification ×20. ( D ) Triple FDC, LLT1, and PNA stain of a frozen tonsillar human section. Interaction between FDCs and double-positive LLT1-PNA GC B cells can be observed (red square). Representative image of six different follicles. Original magnification ×63 oil objective with 3× digital zoom.

Article Snippet: For LLT1 crosslinking, recombinant CD161 (R&D Systems) or IgG1 isotype control (R&D Systems) were bound to a 96-well ELISA plate (Greiner Bio-One, Stonehouse, U.K.) overnight prior to the addition of B cells and BCR stimulus as described above.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Marker

Journal: The Journal of Immunology Author Choice

Article Title: LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation

doi: 10.4049/jimmunol.1502462

Figure Lengend Snippet: LLT1 promotes B cell activation. ( A ) Purified B cells from PBMCs were stimulated with anti-BCRs only or with anti-BCRs with and without rCD161 or IC. Geometric mean fluorescence intensities (GeoMFI) of CD83 and CD38 were measured by flow cytometry. B cell stimulation with rCD161 resulted in enhanced levels of CD83 and CD38; data were pooled from three independent experiments ( n = 7, ** p < 0.01). ( B ) Tonsillar cells were cultured for 3 d with CpG or anti-CD40 plus IL-4. Blocking LLT1–CD161 interaction resulted in a decreased expression of CD83 and CD38 on B cells; data were pooled from four independent experiments ( n = 12, **** p < 0.0001). ( C ) Depletion of CD161 + cells from tonsillar cells resulted in decreased expression of CD83 (anti-CD40 plus IL-4 stimulation) and CD38 (CpG stimulation) on B cells after 3 d; data were pooled from two independent experiments ( n = 8; * p < 0.05, **** p < 0.0001, paired t test). ( D ) Overnight stimulation of tonsillar B cells with rCD161 or 2H7 anti-LLT1 Ab did not change the expression of CD83 on GC B cells ( n = 5). ( E ) However, a decrease in CXCR4 expression levels was observed GC B cells ( n = 10; ** p < 0.01, **** p < 0.0001). ( F ) Repeated experiments on sorted DZ B cells revealed a downregulation of CXCR4 mRNA. All statistics were two-way ANOVA using a Bonferroni multiple comparisons test, unless stated otherwise.

Article Snippet: For LLT1 crosslinking, recombinant CD161 (R&D Systems) or IgG1 isotype control (R&D Systems) were bound to a 96-well ELISA plate (Greiner Bio-One, Stonehouse, U.K.) overnight prior to the addition of B cells and BCR stimulus as described above.

Techniques: Activation Assay, Purification, Fluorescence, Flow Cytometry, Cell Stimulation, Cell Culture, Blocking Assay, Expressing