brl37344  (Santa Cruz Biotechnology)

Journal: Frontiers in Immunology

Article Title: Enrichment of novel CD3+F4/80+ cells in brown adipose tissue following adrenergic stimulation

doi: 10.3389/fimmu.2024.1455407

Figure Lengend Snippet: Enrichment of CD3+F4/80+ DE cells in adrenergically stimulated iBAT SVF. iBAT SVF were collected from mice treated with the β3-adrenergic agonist BRL37344 (10mg/kg) for 4 days. (A) Gating strategy to distinguish CD3+F4/80+ DE cells from T cells (CD3+ F4/80-) and macrophages (Mφ) (CD3-F4/80+) in iBAT SVF. Single, live, CD45+ hematopoietic cells were gated, and CD3+F4/80+ DE, CD3+ F4/80- T cells, and CD3-F4/80+ Mφ were identified. Representative FACS density plots indicate proportions of CD3+F4/80+ DE, CD3+ F4/80- T cells, and CD3-F4/80+ Mφ within CD45+ hematopoietic cells. The frequencies of T cells, macrophages, and DE cells are plotted as a percentage of the total live iBAT SVF cells. (B) Characterization of T cells, macrophages, and CD3+F4/80+ DE cells based on the surface expression of CD4, CD8 (top panels), and TCRβ (bottom panels). CD3+F4/80+ DE cells expressed both CD4 and CD8 but lacked TCRβ expression. Each data point represents an individual mouse. Data are presented as mean ± SD (n = 5). * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001.

Article Snippet: The ß3-adrenergic receptor agonist BRL37344 (Tocris Bioscience, Minneapolis, MN) or PBS was administered intraperitoneally to mice at a dose of 10 mg/kg body weight for four consecutive days.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Enrichment of novel CD3+F4/80+ cells in brown adipose tissue following adrenergic stimulation

doi: 10.3389/fimmu.2024.1455407

Figure Lengend Snippet: Defining immunophenotype of CD3+F4/80+ DE cells with T/NK cells-related markers. (A) A representative FSC/SSC plot (top) and tSNE flow cytometry plot (bottom) displaying the color-coded subsets for CD45+ subsets of iBAT SVF from BRL37344-treated mice. The gating strategy for CD45+ subsets in iBAT SVF is described in Figure 1 . The ungated population (yellow) remains unidentified by these definitions. CD45+ subsets were overlaid onto a tSNE plot, and clustered on CD45+ cells of adrenergically stimulated iBAT SVF. The tSNE flow cytometry plot was based on the surface expression of markers including CD45, CD3, CD4, CD8, CD62L, CD44, CD25, CD127, CD69, PD1, NK1.1, and F4/80. (B) Color scaling of the tSNE plots visualizing the relative surface expression levels of CD45.2, CD3, CD4, CD8, TCRβ and F4/80 in iBAT SVF CD45+ cells. (C) Expression levels of T cell or NK cell-associated markers in CD3+F4/80+ DE cells (red), CD4+ SP T cells (green), CD8+ SP T cells (cyan), and macrophages (blue). Representative histograms (top panels) and quantification of surface marker expression (measured as MFI) for each population. Each data point represents an individual mouse. Data are shown as mean ± SD (n = 5). * P ≤ 0.05, **** P ≤ 0.0001.

Article Snippet: The ß3-adrenergic receptor agonist BRL37344 (Tocris Bioscience, Minneapolis, MN) or PBS was administered intraperitoneally to mice at a dose of 10 mg/kg body weight for four consecutive days.

Techniques: Flow Cytometry, Expressing, Marker

Journal: Investigative Ophthalmology & Visual Science

Article Title: Agonism of β3-Adrenoceptors Inhibits Pathological Retinal Angiogenesis in the Model of Oxygen-Induced Retinopathy

doi: 10.1167/iovs.65.10.34

Figure Lengend Snippet: BRL37344 promotes central retina revascularization and reduces the neovascular tuft area. ( A – D ) Representative images of isolectin B 4 –labeled superficial plexuses of retinas from mice that were normoxic, OIR untreated, or treated with BRL37344 either alone or in combination with the β3-AR antagonist SR59230A. S cale bar : 1 mm. ( a – d ) High magnification of the boxed areas in A to D . S cale bar : 500 µm. The extent of both the avascular area ( D ) and the tuft area ( E ) was quantitatively evaluated in OIR mice untreated or treated with BRL37344 either alone or in combination with SR59230A. Data are shown as box plots with minimum to maximum whiskers ( n = 6 retinas). * P < 0.05 versus normoxic.

Article Snippet: BRL37344 (Sigma-Aldrich, St. Louis, MO, USA), a preferential β3-AR agonist, and SR59230A (Sigma-Aldrich), a β3-AR antagonist, were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) diluted in sterile saline water (at a maximal concentration less than 0.1%) as a vehicle.

Techniques: Labeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Agonism of β3-Adrenoceptors Inhibits Pathological Retinal Angiogenesis in the Model of Oxygen-Induced Retinopathy

doi: 10.1167/iovs.65.10.34

Figure Lengend Snippet: BRL37344 affects hypoxia-driven angiogenic markers. ( A ) Representative western blots and ( B – G ) relative densitometric analysis of HIF-1α, VEGF, PEDF, nNOS, eNOS, and iNOS protein levels in retinal homogenates from normoxic and OIR mice untreated or treated with BRL37344. β-Actin was used as the loading control. Data are shown as box plots with minimum to maximum whiskers ( n = 6 samples). * P < 0.05 versus normoxic; § P < 0.05 versus OIR.

Article Snippet: BRL37344 (Sigma-Aldrich, St. Louis, MO, USA), a preferential β3-AR agonist, and SR59230A (Sigma-Aldrich), a β3-AR antagonist, were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) diluted in sterile saline water (at a maximal concentration less than 0.1%) as a vehicle.

Techniques: Western Blot, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Agonism of β3-Adrenoceptors Inhibits Pathological Retinal Angiogenesis in the Model of Oxygen-Induced Retinopathy

doi: 10.1167/iovs.65.10.34

Figure Lengend Snippet: BRL37344 efficacy on hypoxia-driven angiogenic markers persists up to PD21. ( A ) Representative western blots and ( B , C ) relative densitometric analysis of HIF-1α and vascular endothelial growth factor A (VEGFA) protein levels in retinal homogenates from normoxic and OIR mice untreated or treated with BRL37344 from PD12 to PD16. Measurements were performed at PD17, PD21, and PD25. β-Actin was used as the loading control. Data are shown as box plots with minimum to maximum whiskers ( n = 6 samples). * P < 0.05 versus normoxic; § P < 0.05 versus OIR.

Article Snippet: BRL37344 (Sigma-Aldrich, St. Louis, MO, USA), a preferential β3-AR agonist, and SR59230A (Sigma-Aldrich), a β3-AR antagonist, were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) diluted in sterile saline water (at a maximal concentration less than 0.1%) as a vehicle.

Techniques: Western Blot, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Agonism of β3-Adrenoceptors Inhibits Pathological Retinal Angiogenesis in the Model of Oxygen-Induced Retinopathy

doi: 10.1167/iovs.65.10.34

Figure Lengend Snippet: BRL37344 prevents OIR-associated loss of tight-junction proteins. Representative western blots ( A ) and relative densitometric analysis of ZO-1 ( B ) and claudin-5 ( C ) protein levels in retinal homogenates from normoxic and OIR mice untreated or treated with BRL37344. β-Actin was used as the loading control. Data are shown as box plots with minimum to maximum whiskers ( n = 6 samples). * P < 0.05 versus normoxic; § P < 0.05 versus OIR.

Article Snippet: BRL37344 (Sigma-Aldrich, St. Louis, MO, USA), a preferential β3-AR agonist, and SR59230A (Sigma-Aldrich), a β3-AR antagonist, were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) diluted in sterile saline water (at a maximal concentration less than 0.1%) as a vehicle.

Techniques: Western Blot, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Agonism of β3-Adrenoceptors Inhibits Pathological Retinal Angiogenesis in the Model of Oxygen-Induced Retinopathy

doi: 10.1167/iovs.65.10.34

Figure Lengend Snippet: BRL37344 prevents ERG dysfunction. ( A ) Representative ERG waveforms recorded at a light intensity of 1 log cd·s/m 2 in normoxic and OIR mice untreated or treated with BRL37344. ( B , C ) Scotopic a-wave ( B ) and b-wave ( C ) amplitudes plotted as a function of increasing light intensity in normoxic mice (red circles and red line) and OIR mice untreated (black circles and black line) or treated with subcutaneous BRL37344 (green squares and green line). In untreated OIR mice, the amplitudes of the a- and b-waves were lower than those in the normoxic controls. Each point represents the mean ± SEM of data from six mice. * P < 0.05 versus normoxic; § P < 0.05 versus OIR.

Article Snippet: BRL37344 (Sigma-Aldrich, St. Louis, MO, USA), a preferential β3-AR agonist, and SR59230A (Sigma-Aldrich), a β3-AR antagonist, were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) diluted in sterile saline water (at a maximal concentration less than 0.1%) as a vehicle.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Agonism of β3-Adrenoceptors Inhibits Pathological Retinal Angiogenesis in the Model of Oxygen-Induced Retinopathy

doi: 10.1167/iovs.65.10.34

Figure Lengend Snippet: BRL37344 recovers astrocytes density in the central retina and attenuates OIR-induced gliosis. ( A – C ) Representative images of wholemount retinas stained with isolectin B 4 from normoxic and OIR mice untreated or treated ( left panel ). Scale bar : 1 mm. Boxed areas correspond to high-magnification images of the central retina showing GFAP-IR (astrocytes in green , Müller cells in red ). Scale bars : 50 µm. The 3D reconstructions and Z -stack projections of GFAP-IR display astrocyte profiles and reactive Müller cells processes, respectively. Scale bars : 50 µm (3D reconstructions) and 70 µm ( Z -stack projections). ( D ) Representative western blots and relative densitometric analysis of protein levels of GFAP in retinal homogenates from normoxic and OIR mice untreated or treated with BRL37344. β-Actin was used as the loading control. Data are shown as box plots with minimum to maximum whiskers ( n = 6 samples). * P < 0.05 versus normoxic; § P < 0.05 versus OIR.

Article Snippet: BRL37344 (Sigma-Aldrich, St. Louis, MO, USA), a preferential β3-AR agonist, and SR59230A (Sigma-Aldrich), a β3-AR antagonist, were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) diluted in sterile saline water (at a maximal concentration less than 0.1%) as a vehicle.

Techniques: Staining, Western Blot, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Agonism of β3-Adrenoceptors Inhibits Pathological Retinal Angiogenesis in the Model of Oxygen-Induced Retinopathy

doi: 10.1167/iovs.65.10.34

Figure Lengend Snippet: BRL37344 increases the levels of astrocyte-associated transcription factors. ( A , B ) Representative western blots and relative densitometric analyses of HIF-2α ( A ) and PAX2 ( B ) in retinal homogenates from normoxic and OIR mice untreated or treated with BRL37344. β-actin was used as loading control. Data are shown as box plots with minimum to maximum whiskers ( n = 6 samples). * P < 0.05 versus normoxic; § P < 0.05 versus OIR.

Article Snippet: BRL37344 (Sigma-Aldrich, St. Louis, MO, USA), a preferential β3-AR agonist, and SR59230A (Sigma-Aldrich), a β3-AR antagonist, were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) diluted in sterile saline water (at a maximal concentration less than 0.1%) as a vehicle.

Techniques: Western Blot, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Agonism of β3-Adrenoceptors Inhibits Pathological Retinal Angiogenesis in the Model of Oxygen-Induced Retinopathy

doi: 10.1167/iovs.65.10.34

Figure Lengend Snippet: BRL37344 attenuates OIR-associated RGC loss. ( A ) Representative images of RBPMS-labeled RGCs in wholemount retinas from normoxic and OIR untreated or treated mice ( left panel ). Scale bar : 1 mm. Boxed areas correspond to high-magnification images of the central retina displaying RBPMS labeling counterstained with isolectin B 4 ( right panel ). Scale bar : 25 µm. ( B ) Quantitative evaluation of RBPMS-labeled cell density. Data are shown as box plots with minimum to maximum whiskers ( n = 6 retinas). * P < 0.05 versus normoxic; § P < 0.05 versus OIR.

Article Snippet: BRL37344 (Sigma-Aldrich, St. Louis, MO, USA), a preferential β3-AR agonist, and SR59230A (Sigma-Aldrich), a β3-AR antagonist, were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) diluted in sterile saline water (at a maximal concentration less than 0.1%) as a vehicle.

Techniques: Labeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Agonism of β3-Adrenoceptors Inhibits Pathological Retinal Angiogenesis in the Model of Oxygen-Induced Retinopathy

doi: 10.1167/iovs.65.10.34

Figure Lengend Snippet: BRL37344 prevents OIR-associated PERG alteration. ( A ) Representative PERG waveforms in normoxic and OIR mice untreated or treated with BRL37344. ( B , C ) Quantitative analysis of N1–P1 and P1–N2 amplitudes. Data are shown as box plots with minimum to maximum whiskers ( n = 6 mice). * P < 0.05 versus normoxic; § P < 0.05 versus OIR.

Article Snippet: BRL37344 (Sigma-Aldrich, St. Louis, MO, USA), a preferential β3-AR agonist, and SR59230A (Sigma-Aldrich), a β3-AR antagonist, were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) diluted in sterile saline water (at a maximal concentration less than 0.1%) as a vehicle.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Agonism of β3-Adrenoceptors Inhibits Pathological Retinal Angiogenesis in the Model of Oxygen-Induced Retinopathy

doi: 10.1167/iovs.65.10.34

Figure Lengend Snippet: BRL37344 restrains the upregulation of HIF-1α and β3-AR over the hypoxic phase. ( A , C ) Representative western blots and relative densitometric analysis of HIF-1α and β3-AR protein levels in normoxic and OIR mice untreated or treated with BRL37344. β-Actin was used as the loading control. ( B ) Levels of β3-AR mRNA as determined by qPCR. Data are shown as box plots with minimum to maximum whiskers ( n = 6 samples). * P < 0.05 versus normoxic; § P < 0.05 versus OIR.

Article Snippet: BRL37344 (Sigma-Aldrich, St. Louis, MO, USA), a preferential β3-AR agonist, and SR59230A (Sigma-Aldrich), a β3-AR antagonist, were dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) diluted in sterile saline water (at a maximal concentration less than 0.1%) as a vehicle.

Techniques: Western Blot, Control

Journal: Communications Biology

Article Title: [ 18 F]F-AraG imaging reveals association between neuroinflammation and brown- and bone marrow adipose tissue

doi: 10.1038/s42003-024-06494-x

Figure Lengend Snippet: a Administration of β3 adrenergic receptor agonist BRL37344 (10 mg/kg) 1 h before imaging led to a high accumulation of [ 18 F]F-AraG in iBAT (white arrowhead). No significant uptake was observed in the insulin-treated or control mice. b Signal in the iBAT of adrenergically stimulated mice (10.98 ± 1.64%ID/g) was significantly higher than the signal in the insulin treated (3.36 ± 0.62%ID/g) and control mice (4.11 ± 0.76%ID/g). No significant differences in iBAT signal were found between insulin-treated and control mice ( p = 0.53). c The baseline 18 FDG uptake in iBAT (white arrowheads) that was observed in control mice was increased in both insulin and BRL37344 treated mice, indicating lack of selectivity for adrenergic stimulation. d Administration of BRL37344 (10 mg/kg) for 4 consecutive days led to increased [ 18 F]F-AraG signal in the intrascapular BAT (white arrowhead) and axillary BAT (red arrow) but also in the lumbar vertebrae region (yellow arrowhead), and the bone marrow of the tibia and femur (orange arrowhead). e – g Signal in the lumbar, thoracic, and cervical vertebrae of chronically stimulated mice was significantly different than the signal in acutely BRL37344- treated, insulin-treated and control mice. ID injected dose. [ 18 F]F-AraG uptake in different region of interest was calculated as %ID/g. Data are plotted as mean ± SD ( n = 5 or 6). Each spot represents an individual animal. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.000.

Article Snippet: For the acute adrenergic stimulation, C57BL/6 mice were intraperitoneally (ip) treated with 10 mg/kg dose of β3 agonist BRL37344 (Tocris Bioscience, Minneapolis, MN) 1 h prior to [ 18 F]F-AraG injection and PET/CT imaging.

Techniques: Imaging, Control, Injection

Journal: Communications Biology

Article Title: [ 18 F]F-AraG imaging reveals association between neuroinflammation and brown- and bone marrow adipose tissue

doi: 10.1038/s42003-024-06494-x

Figure Lengend Snippet: a Chronic adrenergic stimulation led to a 6-fold increase in the number of adipocytes in the iBAT. b Following treatment with adrenergic agonist, mRNA expression of markers of activation and thermogenesis were increased in iBAT. Expression of each gene was normalized against 18S expression level. Relative expression levels are presented as fold induction over PBS control mice. c Increases in both CD4+ and CD8+ population contributed to the increase in T cells in iBAT post treatment with BRL37344. d Adrenergic agonist led to the change in subset composition of CD4+ T cells, with naïve cells increasing in frequency. e Following adrenergic stimulation, proportion of non-hematopoietic population increased in the femoral bone marrow. f Expression of Cidea , a briteness marker, increased in the bone marrow post adrenergic treatment, while the expression of other thermogenesis-related genes remained the same. g Representative flow cytometry plots show an increase in lineage-negative mitochondria-rich population in femur bone marrow following adrenergic stimulation. h Majority of mitochondria-rich cells were positive for lipid droplets staining with Nile Red dye. i Post adrenergic stimulation, overwhelming majority of mitochondria-rich population in the bone marrow contained lipid droplets, while T cells represented only a negligible portion. j Treatment with BRL37344 did not significantly change the proportion of T cells in femur bone marrow. k In contrast to iBAT, no significant changes were noted within the CD4+ population in the bone marrow after adrenergic stimulation. l In the bone marrow, frequency of CD4+ cells expressing retention marker CD69 and exhaustion/activation marker PD-1 increased post administration of the adrenergic agonist. m The graph shows median fluorescence intensities (MFI) of MitoTracker Green of femur bone marrow T cells. Following adrenergic stimulation, mitochondrial content of bone marrow T cells decreased ( p = 0.004), suggesting immunosuppression. Data are shown as mean ± SD ( n = 5). Each spot represents an individual mouse. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p < 0.0001.

Article Snippet: For the acute adrenergic stimulation, C57BL/6 mice were intraperitoneally (ip) treated with 10 mg/kg dose of β3 agonist BRL37344 (Tocris Bioscience, Minneapolis, MN) 1 h prior to [ 18 F]F-AraG injection and PET/CT imaging.

Techniques: Expressing, Activation Assay, Control, Marker, Flow Cytometry, Staining, Fluorescence

Journal: Communications Biology

Article Title: [ 18 F]F-AraG imaging reveals association between neuroinflammation and brown- and bone marrow adipose tissue

doi: 10.1038/s42003-024-06494-x

Figure Lengend Snippet: a Chronic adrenergic stimulation of thermogenesis-deficient Letmd1 -KO mice did not result in increased [ 18 F]F-AraG uptake in either iBAT or BMAT. b BRL37344 treatment did not lead to a significant increase in [ 18 F]F-AraG accumulation in the iBAT ( p = 0.08) or lumbar BMAT ( p = 0.14). Signal in the cervical vertebrae increased marginally (12.5%) post adrenergic stimulation ( p = 0.02). c There was no increase in the abundance of adipocyte or lymphocyte in the femoral bone marrow of adrenergically stimulated Letmd1 KO mice. Frequency of the subset is shown. d In Letmd1 -KO mice, the adipocyte population in the femoral bone marrow constituted the majority of cells with a high mitochondrial content. BRL37344 did not change mitochondrial content of femur bone marrow adipocytes. e In femur bone marrow, adrenergic stimulation of Letmd1 -KO mice decreased the population of adipocytes rich in mitochondria. f In T cell-deficient Rag1 -KO mice, treatment with BRL37344 resulted in increased accumulation of [ 18 F]F-AraG in iBAT, whereas there was no notable change in BMAT. g After adrenergic stimulation, [ 18 F]F-AraG signal in the iBAT increased by close to threefold ( p = 0.002), while the signal in BMAT did not show significant changes. h The frequency of adipocytes in the bone marrow of Rag1 -KO mice increased significantly post adrenergic stimulation ( p = 0.01). Frequency of the subset is depicted. i . Rag1 deficiency reduced mitochondrial content in femur bone marrow adipocytes. j The proportion of mitochondria-rich bone marrow adipocytes remained unchanged in adrenergically stimulated Rag1 -KO mice ( p = 0.75). k In wt mice, 18 FDG accumulated in the adrenergically stimulated iBAT but not in the BMAT. Data are shown as mean ± SD ( n = 5). Each spot represents an individual mouse. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p < 0.0001.

Article Snippet: For the acute adrenergic stimulation, C57BL/6 mice were intraperitoneally (ip) treated with 10 mg/kg dose of β3 agonist BRL37344 (Tocris Bioscience, Minneapolis, MN) 1 h prior to [ 18 F]F-AraG injection and PET/CT imaging.

Techniques: