Journal:

Article Title: The Role of Hepatic Invariant (I)NKT Cells in Systemic/Local Inflammation and Mortality During Polymicrobial Septic Shock 1

doi: 10.4049/jimmunol.0801463

Figure Lengend Snippet: Ex vivo intracellular cytokine staining of isolated liver NKT cells (as delineated by CD3+NK1.1+) (A-E) or liver macrophages / Kupffer cells (as delineated by F4/80+) (F-H), derived from sham or CLP C57BL/6J mice, exhibited a marked increase in the frequency of cells expressing the TNF-α, IL-6 as well as IL-10 at 24 h post-CLP. Results are given as mean ± SEM. *, indicates significance at p<0.05 vs. the Sham group; n=5-7 mice/group.

Article Snippet: NPC were stained with PE-conjugated anti-CD3, anti-CD4, anti-CD69, anti-CD25, anti-CD1d antibodies (BD-Biosciences Inc) and APC-conjugated anti-F4/80, anti-CD11c, anti-NK1.1 antibodies (BD-Biosciences Inc), α-galactosylceramide-loaded (α-GalCer) as well as unloaded CD1d tetramer (provided by the tetramer facility at the National Institute of Health, Germantown, MD) and with the appropriate antibody isotypic-chromaphore conjugated controls (BD-Biosciences Inc).

Techniques: Ex Vivo, Staining, Isolation, Derivative Assay, Expressing

Journal:

Article Title: The Role of Hepatic Invariant (I)NKT Cells in Systemic/Local Inflammation and Mortality During Polymicrobial Septic Shock 1

doi: 10.4049/jimmunol.0801463

Figure Lengend Snippet: Assessment of the effect of in vivo anti-CD1d antibody pre-treatment (as opposed to non-specific mouse IgG treatment) of CLP mice on the ex vivo intracellular cytokine staining of isolated liver NKT cells (as delineated by CD3+DX5+) (A-E) or liver macrophage/ Kupffer cells (as delineated by F4/80+) (F-H), derived from Balb/c mice. Liver NKT cells exhibited a marked increase only in the frequency of cells expressing the IL-4 and IL-10 but not IFN-γ, TNF-α, IL-6 or at 24 h post-CLP that was partially blocked by anti-CD1d treatment. (A-E). Alternatively, CLP/IgG treated mouse liver macrophages show an increase in the percentage of TNF-α+ and IL-6+ cells at 24 h post-CLP. Only the increased IL-6 production was decreased by pre-treatment with anti-CD1d antibodies. (F-H). Results are given as mean ± SEM. *, indicates significance at p<0.05 vs. the Sham group; #, indicates significance at p<0.05 vs. the CLP-Sham group; n=3-5 mice/group.

Article Snippet: NPC were stained with PE-conjugated anti-CD3, anti-CD4, anti-CD69, anti-CD25, anti-CD1d antibodies (BD-Biosciences Inc) and APC-conjugated anti-F4/80, anti-CD11c, anti-NK1.1 antibodies (BD-Biosciences Inc), α-galactosylceramide-loaded (α-GalCer) as well as unloaded CD1d tetramer (provided by the tetramer facility at the National Institute of Health, Germantown, MD) and with the appropriate antibody isotypic-chromaphore conjugated controls (BD-Biosciences Inc).

Techniques: In Vivo, Ex Vivo, Staining, Isolation, Derivative Assay, Expressing

Journal: Cell Reports Medicine

Article Title: Commensal bacteria promote azathioprine therapy failure in inflammatory bowel disease via decreasing 6-mercaptopurine bioavailability

doi: 10.1016/j.xcrm.2023.101153

Figure Lengend Snippet:

Article Snippet: Ms F4/80 APC-R700 T45-2342 , BD Pharmingen , Cat# 565787; RRID: AB_2869711.

Techniques: Staining, Recombinant, Selection, Activity Assay, Multiplex Assay, Immunohistochemical staining, Sequencing, Software

Journal: bioRxiv

Article Title: Scarless repair of acute and chronic kidney injury in African Spiny mice ( Acomys cahirinus )

doi: 10.1101/315069

Figure Lengend Snippet: (A) Smooth muscle alpha actin ( Acta2 ) expression was investigated by immune-confocal microscopy at days 3, 7, and 14 after UUO. Representative digital images (400x) Acta2 expression (green) for B6 and A. cahirinus at days 7 and 14 after UUO; (B) Graph summarizes image analysis for Acta2 at each time point (n=6/time point for each group). (C) F4/80 macrophage infiltration was examined by confocal microscopy and quantified by flow cytometry. Representative digital images (400x) of F4/80 (red) expression at days 7 and 14 after UUO. (D) Flow cytometry was performed on single cell suspensions from whole kidney and analyzed for F4/80 expression. Graph summarizes percent F4/80+ cells at days 3, 7, and 14 after UUO analyzed by FACS (n=3-4/time point per group). (E) Tubular integrity was examined by confocal microscopy for Cdh1 (E cadherin). Representative digital images (400x) of Cdh1 (red) expression for B6 and A. cahirinus . (F) Graph summarizes image analysis results for Cdh1 levels at each time point (n=6/time point for each group). (B6 (blue) vs. A. cahirinus (red): *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; between time points # p<0.05, ## p<0.01)

Article Snippet: Cells were stained per protocol with DAPI, PE-Cy7-anti-CD45, PE-anti-CD11b, APC-eFluor780-F4/80 from BD Sciences.

Techniques: Expressing, Confocal Microscopy, Flow Cytometry

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Polydatin radiosensitizes lung cancer while preventing radiation injuries by modulating tumor-infiltrating B cells

doi: 10.1007/s00432-023-04762-7

Figure Lengend Snippet: Tumor-associated B cells analysis in vivo. a , b Representative histograms of each representative treatment group in flow cytometry experiments. c , d Quantitative comparison of the B220- or CD19-positive rates between different treatment groups calculated from three independent flow cytometry tests. e , f Representative pictures and the quantitative statistical histogram for the CD19 IHC experiment for labeling B cells within the tumors. g Mechanism diagram for PD’s RMDF role in lung cancer radiotherapy. * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: Subsequently, 1 × 10 5 cells in 100 μl were stained for surface markers with the following antibodies: PE-conjugated CD19 (Biogems), APC-conjugated B220 (Biogems), and FITC-conjugated rabbit IgM (Biogems).

Techniques: In Vivo, Flow Cytometry, Comparison, Labeling

Journal: JCI Insight

Article Title: Angiotensin-(1-7)/MasR axis promotes migration of monocytes/macrophages with a regulatory phenotype to perform phagocytosis and efferocytosis

doi: 10.1172/jci.insight.147819

Figure Lengend Snippet: Briefly, BALB/c mice received an i.pl. injection of Ang-(1-7) (100 ng/cavity) or PBS (controls), and the macrophages recruited to the cavity were harvested at 48 hours for phenotyping by flow cytometry as shown in gating strategy ( A ). ( B ) Graphs present the absolute numbers of M1 (F4/80 lo Ly6C + CD11b med ), M2 (F4/80 hi Ly6C – CD11b hi ), and Mres (F4/80 med CD11b lo ) recruited into the pleura. TGF-β levels were assessed in the pleural lavage supernatant from Ang-(1-7)–injected mice at different time points postinjection ( C ). Leukocytes recruited into the pleural cavity were processed for Western blot analysis of Arg-1 and Ym1 levels ( D ). β-Actin was used as a loading control. During in vitro settings, the kinetics of production of IL-10 and TGF-β by BMDMs were evaluated ( E ). Data are presented as mean ± SEM of 8 mice per group (in vivo) or are representative results of 3 independent experiments with BMDMs performed in biological quadruplicates ( n = 4). Western blot quantification was performed using ImageJ software from the representative blots shown in D , which used whole cell extracts from 3 mice. * for P < 0.05 and *** for P < 0.001 when compared with the control group (PBS) by t test ( B ) or 1-way ANOVA ( C and E ).

Article Snippet: Primary antibodies — PE-conjugated anti-CCR2 (LS132.1D9 1:50 — R&D Systems), APC-conjugated rat anti-F4/80 (T45-2342 1:50 — BD Biosciences), and rabbit anti-MasR (MAS1L ab200685 1:200 — Abcam) — were incubated overnight at 4°C.

Techniques: Injection, Flow Cytometry, Western Blot, In Vitro, In Vivo, Software

Journal: JCI Insight

Article Title: Angiotensin-(1-7)/MasR axis promotes migration of monocytes/macrophages with a regulatory phenotype to perform phagocytosis and efferocytosis

doi: 10.1172/jci.insight.147819

Figure Lengend Snippet: BALB/c mice were challenged with LPS (250 ng/cavity, i.pl.) or PBS, and leukocytes from the pleural cavity were harvested after 8, 24, and 48 hours for Western blot analysis of MasR ( A ) and differential cell counts ( B and C ). LPS-challenged mice were treated with A779 (200 ng/cavity) or vehicle at 8 and 24 hours post-LPS injection, and leukocytes were harvested at 48 hours for differential cell counts ( B and C ). Next, WT and MasR –/– mice were also i.pl. challenged with LPS, and neutrophil ( D ) and mononuclear cell numbers ( E ) and CCL2 levels ( F ) were evaluated. Flow cytometry analysis was performed to assess numbers of macrophages (F4/80 + CD11b + — G ), monocytes (F4/80 – Ly6C + — H ), and neutrophils (F4/80 – Ly6G + — I ). Frequencies of CD206 + macrophages are graphed in J and representative gating is shown in K . Results are shown as the mean ± SEM of n = 5–6 mice. * for P < 0.05, ** for P < 0.01, and *** for P < 0.001 when compared with the control group (PBS). ## for P < 0.01 when compared with the 8-hour time point, or as indicated, by 1-way ANOVA ( B and C ) or 2-way ANOVA ( D – K ). NSB, nonspecific band.

Article Snippet: Primary antibodies — PE-conjugated anti-CCR2 (LS132.1D9 1:50 — R&D Systems), APC-conjugated rat anti-F4/80 (T45-2342 1:50 — BD Biosciences), and rabbit anti-MasR (MAS1L ab200685 1:200 — Abcam) — were incubated overnight at 4°C.

Techniques: Western Blot, Injection, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: TREM2 expression promotes liver and peritoneal M2 macrophage polarization in mice infected with Schistosoma japonicum

doi: 10.1111/jcmm.17842

Figure Lengend Snippet: Trem2 deletion increases F4/80 + CD86+ cells in peritoneal macrophages. (A) TREM2 expression is upregulated in peritoneal macrophages from mice infected with Schistosoma japonicum . The number of TREM2+ cells in F4/80 + CD11b + peritoneal macrophages was determined by flow cytometry and compared to uninfected controls. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) The number of F4/80 + CD86+ cells in total peritoneal macrophages obtained from wild‐type or Trem2 −/− mice with or without IL4/13 stimulation was determined by flow cytometry. ** p < 0.01. (C) The number of F4/80 + CD206+ cells in total peritoneal macrophages obtained from wild‐type or Trem2 −/− mice with or without IL4/13 stimulation was determined by flow cytometry. * p < 0.05. All results are relative to their respective controls.

Article Snippet: For this, the cells were incubated with PE conjugated antibody against mouse F4/80 (02922–60, Biogems), PE‐Cy7 conjugated antibody against mouse CD11b (03221–77, Biogems), APC conjugated antibody against mouse CD86 or CD206 (17–0862, 17–2061, eBioscience) and FITC conjugated antibody against mouse TREM2 (ab119852, Abcam).

Techniques: Expressing, Infection, Flow Cytometry