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bodipy-fl-c16 d3821 (Thermo Fisher)

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Thermo Fisher bodipy-fl-c16 d3821
Bodipy Fl C16 D3821, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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bodipy-fl-c16 d3821 - by Bioz Stars, 2026-02

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a , b Representative ORO staining and quantitative analysis of the whole aorta from 8-week-old ApoE KO mice fed a normal chow (NC) or a high-fat diet (HFD) for 0, 8, and 16 weeks ( n = 4 mice). Scale bar, 5 mm. c En face immunofluorescence images of the inner curvature of the aortic arch VECs stained with antibodies against VE-cadherin (white) and ac-tubulin (magenta). LDs were stained with BODIPY (green), and nuclei were stained with DAPI (blue). Scale bar, 10 μm. d , e Quantification of BODIPY staining ( d ) and ciliation ( e ) of VECs of the aortic arch shown in ( c ) ( n = 10 mice). f , g Scatter plots showing the relationship between ciliary signal and LD signal of randomly chosen ciliated VECs of the inner curvature of the aortic arch ( n = 10 mice for each group). Male mice were used for this study. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with post hoc analysis. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Lipid droplets sequester palmitic acid to disrupt endothelial ciliation and exacerbate atherosclerosis in male mice

doi: 10.1038/s41467-024-52621-x

Figure Lengend Snippet: a , b Representative ORO staining and quantitative analysis of the whole aorta from 8-week-old ApoE KO mice fed a normal chow (NC) or a high-fat diet (HFD) for 0, 8, and 16 weeks ( n = 4 mice). Scale bar, 5 mm. c En face immunofluorescence images of the inner curvature of the aortic arch VECs stained with antibodies against VE-cadherin (white) and ac-tubulin (magenta). LDs were stained with BODIPY (green), and nuclei were stained with DAPI (blue). Scale bar, 10 μm. d , e Quantification of BODIPY staining ( d ) and ciliation ( e ) of VECs of the aortic arch shown in ( c ) ( n = 10 mice). f , g Scatter plots showing the relationship between ciliary signal and LD signal of randomly chosen ciliated VECs of the inner curvature of the aortic arch ( n = 10 mice for each group). Male mice were used for this study. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with post hoc analysis. Source data are provided as a Source Data file.

Article Snippet: BODIPY TM 493/503 (D3922), BODIPY TM FL C16 (D3821), HCS LipidTOX (H34477), DAPI (D3571), Protein A/G Agarose (20421), Streptavidin Agarose beads (88817), and BMCC-Biotin (21900) were purchased from Thermo Fisher.

Techniques: Staining, Immunofluorescence

$a Metabolic pathway of indicated fatty acids. SCD1 stearoyl-CoA desaturase-1, Elovl Elongation of very long-chain fatty acid, Δ6D Δ6 desaturase, PA palmitic acid, POA palmitoleic acid, SA stearic acid, OA oleic acid, LA linoleic acid, GLA gamma-linolenic acid, DGLA dihomo-γ-linolenic acid, AA arachidonic acid. b , c GC-MS analysis showing changes in levels of indicated free fatty acids in HUVECs ( n = 6 samples). ELA elaidic acid, EA erucic acid. d GC-MS analysis showing changes in the total free fatty acid level in HUVECs ( n = 6 samples). e – g Immunofluorescence images ( e ) and quantifications of LipidTOX staining ( f ) and cytosolic BODIPY FL C16 signal ( g ) of treated HUVECs ( n = 10 fields from 3 independent experiments). Scale bar, 5 µm. h – j LC-MS analysis showing the fractional abundance of 13 C 16 -labeled PA in the cytosol ( h ), 13 C 16 -labeled triglycerides in whole cells ( i ), and 13 C 16 -labeled PA in the culture medium ( j ) of HUVECs with the following treatment: OA (200 μM) for 12 h; DGAT1 overexpression for 48 h; or Atglistatin (10 µM) for 24 h ( n = 3 samples). HUVECs were pretreated with 13 C 16 -PA (200 μM) for 24 h and rinsed with fresh medium before the above treatments. k – p Immunofluorescence images ( k , n ) and quantifications of LipidTOX staining ( l , o ) and ciliation ( m , p ) of HAECs treated with 200 μM OA and/or 200 μM SA or PA for 12 h ( n = 20 fields from 3 independent experiments). Cells were treated with BSA or OA for 12 h, exposed to SA or PA for 12 h, and then serum-starved for 48 h. Scale bars, 20 µm. Data are presented as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t -test ( b , c , h , j ), one-way ( d ), or two-way ( f , g , l , m , o , and p ) ANOVA with post hoc analysis. Source data are provided as a Source Data file.$

Journal: Nature Communications

Article Title: Lipid droplets sequester palmitic acid to disrupt endothelial ciliation and exacerbate atherosclerosis in male mice

doi: 10.1038/s41467-024-52621-x

Figure Lengend Snippet: a Metabolic pathway of indicated fatty acids. SCD1 stearoyl-CoA desaturase-1, Elovl Elongation of very long-chain fatty acid, Δ6D Δ6 desaturase, PA palmitic acid, POA palmitoleic acid, SA stearic acid, OA oleic acid, LA linoleic acid, GLA gamma-linolenic acid, DGLA dihomo-γ-linolenic acid, AA arachidonic acid. b , c GC-MS analysis showing changes in levels of indicated free fatty acids in HUVECs ( n = 6 samples). ELA elaidic acid, EA erucic acid. d GC-MS analysis showing changes in the total free fatty acid level in HUVECs ( n = 6 samples). e – g Immunofluorescence images ( e ) and quantifications of LipidTOX staining ( f ) and cytosolic BODIPY FL C16 signal ( g ) of treated HUVECs ( n = 10 fields from 3 independent experiments). Scale bar, 5 µm. h – j LC-MS analysis showing the fractional abundance of 13 C 16 -labeled PA in the cytosol ( h ), 13 C 16 -labeled triglycerides in whole cells ( i ), and 13 C 16 -labeled PA in the culture medium ( j ) of HUVECs with the following treatment: OA (200 μM) for 12 h; DGAT1 overexpression for 48 h; or Atglistatin (10 µM) for 24 h ( n = 3 samples). HUVECs were pretreated with 13 C 16 -PA (200 μM) for 24 h and rinsed with fresh medium before the above treatments. k – p Immunofluorescence images ( k , n ) and quantifications of LipidTOX staining ( l , o ) and ciliation ( m , p ) of HAECs treated with 200 μM OA and/or 200 μM SA or PA for 12 h ( n = 20 fields from 3 independent experiments). Cells were treated with BSA or OA for 12 h, exposed to SA or PA for 12 h, and then serum-starved for 48 h. Scale bars, 20 µm. Data are presented as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t -test ( b , c , h , j ), one-way ( d ), or two-way ( f , g , l , m , o , and p ) ANOVA with post hoc analysis. Source data are provided as a Source Data file.

Techniques: Gas Chromatography-Mass Spectrometry, Immunofluorescence, Staining, Liquid Chromatography with Mass Spectroscopy, Labeling, Over Expression, Two Tailed Test

a Schematic of the reaction catalyzed by SCD1. b The mRNA level of SCD1 in MAECs isolated from ApoE KO mice fed NC or HFD ( n = 6 mice). c , d Immunoblotting ( c ) and quantification ( d , n = 3 mice) of the protein level of SCD1 in MAECs isolated from ApoE KO mice fed NC or HFD. e Levels of indicated fatty acids in HUVECs with or without SCD1 overexpression ( n = 6 samples). f – h Immunofluorescence images ( f ) and quantifications of BODIPY staining ( g ) and ciliation ( h ) of HUVECs treated with palmitic acid (PA) and palmitoleic acid (POA) at the indicated concentration for 12 h, followed by serum starvation for 48 h ( n = 10 fields from 3 independent experiments). Scale bar, 10 µm. i – k Immunofluorescence images ( i ) and quantifications of LipidTOX staining ( j ) and ciliation ( k ) of BSA- or PA (200 µM)-treated HAECs with or without SCD1 overexpression ( n = 20 fields from 3 independent experiments). Boxed areas are enlarged in the bottom panel. Scale bar (for enlarged images), 10 µm. l HUVECs with or without SCD1 overexpression were treated with BSA or PA (200 µM) for 12 h. IP-ABE and immunoblotting were performed to determine the level of ARL13B S-palmitoylation. pcDNA3.1 vector was used as the Vector control. m , n HUVECs with or without SCD1 overexpression were treated with or without PA (200 µM) for 12 h. 20 mg/mL cycloheximide (CHX) was then added for the indicated time. The levels of ARL13B and SCD1 were examined by immunoblotting ( m ) and quantified by densitometry ( n ) ( n = 3 samples). Data are presented as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t -test ( b , d , and e ), one-way ( g , h , n ), or two-way ( j , k ) ANOVA with post hoc analysis. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Lipid droplets sequester palmitic acid to disrupt endothelial ciliation and exacerbate atherosclerosis in male mice

doi: 10.1038/s41467-024-52621-x

Figure Lengend Snippet: a Schematic of the reaction catalyzed by SCD1. b The mRNA level of SCD1 in MAECs isolated from ApoE KO mice fed NC or HFD ( n = 6 mice). c , d Immunoblotting ( c ) and quantification ( d , n = 3 mice) of the protein level of SCD1 in MAECs isolated from ApoE KO mice fed NC or HFD. e Levels of indicated fatty acids in HUVECs with or without SCD1 overexpression ( n = 6 samples). f – h Immunofluorescence images ( f ) and quantifications of BODIPY staining ( g ) and ciliation ( h ) of HUVECs treated with palmitic acid (PA) and palmitoleic acid (POA) at the indicated concentration for 12 h, followed by serum starvation for 48 h ( n = 10 fields from 3 independent experiments). Scale bar, 10 µm. i – k Immunofluorescence images ( i ) and quantifications of LipidTOX staining ( j ) and ciliation ( k ) of BSA- or PA (200 µM)-treated HAECs with or without SCD1 overexpression ( n = 20 fields from 3 independent experiments). Boxed areas are enlarged in the bottom panel. Scale bar (for enlarged images), 10 µm. l HUVECs with or without SCD1 overexpression were treated with BSA or PA (200 µM) for 12 h. IP-ABE and immunoblotting were performed to determine the level of ARL13B S-palmitoylation. pcDNA3.1 vector was used as the Vector control. m , n HUVECs with or without SCD1 overexpression were treated with or without PA (200 µM) for 12 h. 20 mg/mL cycloheximide (CHX) was then added for the indicated time. The levels of ARL13B and SCD1 were examined by immunoblotting ( m ) and quantified by densitometry ( n ) ( n = 3 samples). Data are presented as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t -test ( b , d , and e ), one-way ( g , h , n ), or two-way ( j , k ) ANOVA with post hoc analysis. Source data are provided as a Source Data file.

Techniques: Isolation, Western Blot, Over Expression, Immunofluorescence, Staining, Concentration Assay, Plasmid Preparation, Control, Two Tailed Test

a – c En face immunofluorescence images ( a ) and quantifications of BODIPY staining ( b ) and ciliation ( c ) of aortic arch VECs from IFT88 EC KO ;ApoE KO and littermate ApoE KO mice intravenously injected with the SCD1 inhibitor A939572 (5 mg/kg body weight/2 days) or vehicle for 4 weeks after an 8-week HFD feeding ( n = 6 mice). Scale bar, 10 μm. d Representative images of Oil Red O (ORO) staining of atherosclerosis lesions on the aorta of IFT88 EC KO ;ApoE KO and littermate ApoE KO mice treated as described in ( a ). Boxed areas are enlarged in the top panel. Scale bar (for enlarged images), 1 mm. e Quantification of atherosclerotic lesions shown in ( d ) ( n = 6 mice). f Representative ORO staining in the aortic root of IFT88 EC KO ;ApoE KO and littermate ApoE KO mice treated as described in ( a ). Scale bar, 500 μm. g Quantification of atherosclerotic lesions shown in ( f ) ( n = 6 mice). h Transcriptional levels of IL1B , IL6 , Tgfb1 , Vcam1 , Sele , Tnfaip3 , and Hmox1 , in mice treated as described in ( a ) ( n = 4 mice). Data are presented as mean ± SEM. Statistical significance was determined by two-way ANOVA with post hoc analysis. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Lipid droplets sequester palmitic acid to disrupt endothelial ciliation and exacerbate atherosclerosis in male mice

doi: 10.1038/s41467-024-52621-x

Figure Lengend Snippet: a – c En face immunofluorescence images ( a ) and quantifications of BODIPY staining ( b ) and ciliation ( c ) of aortic arch VECs from IFT88 EC KO ;ApoE KO and littermate ApoE KO mice intravenously injected with the SCD1 inhibitor A939572 (5 mg/kg body weight/2 days) or vehicle for 4 weeks after an 8-week HFD feeding ( n = 6 mice). Scale bar, 10 μm. d Representative images of Oil Red O (ORO) staining of atherosclerosis lesions on the aorta of IFT88 EC KO ;ApoE KO and littermate ApoE KO mice treated as described in ( a ). Boxed areas are enlarged in the top panel. Scale bar (for enlarged images), 1 mm. e Quantification of atherosclerotic lesions shown in ( d ) ( n = 6 mice). f Representative ORO staining in the aortic root of IFT88 EC KO ;ApoE KO and littermate ApoE KO mice treated as described in ( a ). Scale bar, 500 μm. g Quantification of atherosclerotic lesions shown in ( f ) ( n = 6 mice). h Transcriptional levels of IL1B , IL6 , Tgfb1 , Vcam1 , Sele , Tnfaip3 , and Hmox1 , in mice treated as described in ( a ) ( n = 4 mice). Data are presented as mean ± SEM. Statistical significance was determined by two-way ANOVA with post hoc analysis. Source data are provided as a Source Data file.

Techniques: Immunofluorescence, Staining, Injection

a Eight-week-old ApoE KO mice were divided into four groups and fed indicated diets for 12 weeks. NC normal chow, HFD high-fat diet, S soybean oil, P palm oil. b , c Serum palmitic acid (PA) level ( b ) and body weight ( c ) of ApoE KO mice treated as described in ( a ) ( n = 6 mice). d – f En face immunofluorescence images ( d ) and quantifications of BODIPY staining ( e ) and ciliation ( f ) of VECs of the aortic arch from ApoE KO mice treated as described in ( a ) ( n = 6 mice). Scale bar, 20 μm. g Oil Red O (ORO) staining showing atherosclerotic lesions in the aortic tree and the aortic root obtained from mice treated as described in ( a ). Boxed areas are enlarged in the top panel. Scale bar (for the middle panel), 2 mm; Scale bar (for the bottom panel), 400 μm. h , i Quantification of atherosclerotic lesions in the aortic tree ( h ) and the aortic root ( i ) shown in ( g ) ( n = 6 mice). j – m Transcriptional levels of IL6 , IL1B , Sele , and iNOS in the aorta of ApoE KO mice treated as described in ( a ) ( n = 4 mice). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with post hoc analysis. Panel ( a ) was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Lipid droplets sequester palmitic acid to disrupt endothelial ciliation and exacerbate atherosclerosis in male mice

doi: 10.1038/s41467-024-52621-x

Figure Lengend Snippet: a Eight-week-old ApoE KO mice were divided into four groups and fed indicated diets for 12 weeks. NC normal chow, HFD high-fat diet, S soybean oil, P palm oil. b , c Serum palmitic acid (PA) level ( b ) and body weight ( c ) of ApoE KO mice treated as described in ( a ) ( n = 6 mice). d – f En face immunofluorescence images ( d ) and quantifications of BODIPY staining ( e ) and ciliation ( f ) of VECs of the aortic arch from ApoE KO mice treated as described in ( a ) ( n = 6 mice). Scale bar, 20 μm. g Oil Red O (ORO) staining showing atherosclerotic lesions in the aortic tree and the aortic root obtained from mice treated as described in ( a ). Boxed areas are enlarged in the top panel. Scale bar (for the middle panel), 2 mm; Scale bar (for the bottom panel), 400 μm. h , i Quantification of atherosclerotic lesions in the aortic tree ( h ) and the aortic root ( i ) shown in ( g ) ( n = 6 mice). j – m Transcriptional levels of IL6 , IL1B , Sele , and iNOS in the aorta of ApoE KO mice treated as described in ( a ) ( n = 4 mice). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with post hoc analysis. Panel ( a ) was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Techniques: Immunofluorescence, Staining

Lipid metabolism dysregulation triggered by protein-energy restriction during M. avium infection . (a) The superior lobes of the right lung were examined through H&E staining in subsets of mice. Representative images from five mice per group at 10 weeks p.i. are displayed (200X: scale bars = 100 μm, 400X: scale bars = 50 μm). (b) Representative flow cytometric plots depict the frequency of BODIPY 493/503 + cells within live cell populations at 10 weeks p.i., alongside bar graphs for quantitative comparison. (c) Principal component analysis conducted on 15 lung tissue samples at 10 weeks p.i. (n = 5 per group). (d) The heat map displays significant differences in TAG, CE, DAG, and FFA levels among various dietary groups in lung tissue at 10 weeks p.i., as determined by one-way ANOVA ( p < 0.05 significance threshold) (n = 5 per group). (e) FFA species in lung tissues at 10 weeks p.i. were analysed using UPLC-Q Exactive/MS. Data are normalised to the SPD-fed group and displayed as scatter plots with bar graphs (n = 5 per group). (f–h) Gene expression analysis in lung tissue at 10 weeks p.i. for lipid metabolism-related enzymes, including Acsl3 , Acsl6 , Dgat1 , Dgat2 (f), Atgl , Hsl (g), and Cd36 (h). mRNA levels were quantified relative to β-actin and normalised to the SPD-fed group (n = 5 per group). The results are presented as the mean ± SD, and statistical significance was determined by unpaired Student's t-test and one-way ANOVA with Tukey's multiple comparison test (NS, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). SPD, standard protein diet; LPD, low protein diet; H&E, hematoxylin and eosin; p.i., post-infection; TAG, triacylglycerol; CE, cholesteryl ester; DAG, diacylglycerol; FFA, free fatty acid; UPLC-Q Exactive/MS, Ultra Performance Liquid Chromatography-Q Exactive Mass Spectrometry; Acsl3 , acyl-CoA synthetase long-chain family member 3; Acsl6 , acyl-CoA synthetase long-chain family member 6; Dgat1 , diacylglycerol O-acyltransferase 1; Dgat2 , diacylglycerol O-acyltransferase 2; Atgl , adipose triglyceride lipase; Hsl , hormone-sensitive lipase.

Journal: eBioMedicine

Article Title: Protein-energy restriction-induced lipid metabolism disruption causes stable-to-progressive disease shift in Mycobacterium avium -infected female mice

doi: 10.1016/j.ebiom.2024.105198

Figure Lengend Snippet: Lipid metabolism dysregulation triggered by protein-energy restriction during M. avium infection . (a) The superior lobes of the right lung were examined through H&E staining in subsets of mice. Representative images from five mice per group at 10 weeks p.i. are displayed (200X: scale bars = 100 μm, 400X: scale bars = 50 μm). (b) Representative flow cytometric plots depict the frequency of BODIPY 493/503 + cells within live cell populations at 10 weeks p.i., alongside bar graphs for quantitative comparison. (c) Principal component analysis conducted on 15 lung tissue samples at 10 weeks p.i. (n = 5 per group). (d) The heat map displays significant differences in TAG, CE, DAG, and FFA levels among various dietary groups in lung tissue at 10 weeks p.i., as determined by one-way ANOVA ( p < 0.05 significance threshold) (n = 5 per group). (e) FFA species in lung tissues at 10 weeks p.i. were analysed using UPLC-Q Exactive/MS. Data are normalised to the SPD-fed group and displayed as scatter plots with bar graphs (n = 5 per group). (f–h) Gene expression analysis in lung tissue at 10 weeks p.i. for lipid metabolism-related enzymes, including Acsl3 , Acsl6 , Dgat1 , Dgat2 (f), Atgl , Hsl (g), and Cd36 (h). mRNA levels were quantified relative to β-actin and normalised to the SPD-fed group (n = 5 per group). The results are presented as the mean ± SD, and statistical significance was determined by unpaired Student's t-test and one-way ANOVA with Tukey's multiple comparison test (NS, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). SPD, standard protein diet; LPD, low protein diet; H&E, hematoxylin and eosin; p.i., post-infection; TAG, triacylglycerol; CE, cholesteryl ester; DAG, diacylglycerol; FFA, free fatty acid; UPLC-Q Exactive/MS, Ultra Performance Liquid Chromatography-Q Exactive Mass Spectrometry; Acsl3 , acyl-CoA synthetase long-chain family member 3; Acsl6 , acyl-CoA synthetase long-chain family member 6; Dgat1 , diacylglycerol O-acyltransferase 1; Dgat2 , diacylglycerol O-acyltransferase 2; Atgl , adipose triglyceride lipase; Hsl , hormone-sensitive lipase.

Article Snippet: BMDMs were seeded at a density of 1 × 10 6 cells/mL on a 60-mm culture plate and incubated for 24 h. After washing with PBS, the cells underwent treatment with SSO and αCD36 for 4 h. Following another PBS wash, BMDMs were exposed to 1 ug/mL of BODIPY FL C16 (#D3821, Thermo Fisher) for 1 h. Cell harvesting was carried out using trypsin–EDTA, and intracellular fluorescence was measured using flow cytometry.

Techniques: Infection, Staining, Comparison, Gene Expression, Liquid Chromatography, Mass Spectrometry

Analysis of serum free fatty acids in patients with MAC-PD at the time of diagnosis . (a) Quantification of total serum FFA levels in patients with MAC-PD and healthy controls. The scatterplot displays individual data points. The results are presented as the median ± IQR and Mann–Whitney rank tests were used for group comparisons (NS, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001). (b) Detailed comparison of individual serum FFA including C16:0, C16:1, C18:0, C18:2, C18:3, and C20:4 using UPLC-Q Exactive/MS. Each scatterplot represents peak area values for individual FFA. The results are presented as the median ± IQR and Mann–Whitney rank tests were used for group comparisons (NS, not significant; ∗∗ p < 0.01). (c) Correlation between BMI and peak area of the C16:0 metabolite in twenty-six patients who showed progression of MAC-PD. A two-tailed Spearman's rank correlation coefficient ( rho ) was calculated (∗ p < 0.05). The linear regression line is presented for illustrative purposes to suggest a potential linear trend. (d) Correlation between BMI and the time from diagnosis to treatment in twenty-six patients who showed progression of MAC-PD. A two-tailed Spearman's rank correlation coefficient ( rho ) was calculated (∗ p < 0.05). The linear regression line is presented for illustrative purposes to suggest a potential linear trend. MAC-PD, Mycobacterium avium complex-pulmonary disease; FFA, free fatty acid; UPLC-Q Exactive/MS, Ultra Performance Liquid Chromatography-Q Exactive Mass Spectrometry; IQR, Interquartile Range.

Journal: eBioMedicine

Article Title: Protein-energy restriction-induced lipid metabolism disruption causes stable-to-progressive disease shift in Mycobacterium avium -infected female mice

doi: 10.1016/j.ebiom.2024.105198

Figure Lengend Snippet: Analysis of serum free fatty acids in patients with MAC-PD at the time of diagnosis . (a) Quantification of total serum FFA levels in patients with MAC-PD and healthy controls. The scatterplot displays individual data points. The results are presented as the median ± IQR and Mann–Whitney rank tests were used for group comparisons (NS, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001). (b) Detailed comparison of individual serum FFA including C16:0, C16:1, C18:0, C18:2, C18:3, and C20:4 using UPLC-Q Exactive/MS. Each scatterplot represents peak area values for individual FFA. The results are presented as the median ± IQR and Mann–Whitney rank tests were used for group comparisons (NS, not significant; ∗∗ p < 0.01). (c) Correlation between BMI and peak area of the C16:0 metabolite in twenty-six patients who showed progression of MAC-PD. A two-tailed Spearman's rank correlation coefficient ( rho ) was calculated (∗ p < 0.05). The linear regression line is presented for illustrative purposes to suggest a potential linear trend. (d) Correlation between BMI and the time from diagnosis to treatment in twenty-six patients who showed progression of MAC-PD. A two-tailed Spearman's rank correlation coefficient ( rho ) was calculated (∗ p < 0.05). The linear regression line is presented for illustrative purposes to suggest a potential linear trend. MAC-PD, Mycobacterium avium complex-pulmonary disease; FFA, free fatty acid; UPLC-Q Exactive/MS, Ultra Performance Liquid Chromatography-Q Exactive Mass Spectrometry; IQR, Interquartile Range.

Techniques: Biomarker Discovery, MANN-WHITNEY, Comparison, Two Tailed Test, Liquid Chromatography, Mass Spectrometry

Enhanced CD36 expression and neutral lipid accumulation in macrophages by protein-energy restriction during M. avium infection . (a) CD36 expression in immune cells in the lung analysed by flow cytometry at 10 weeks p.i. The scatter plots with bar graphs depict the MFI of CD36 expression. Fluorescence minus one control was used to define the negative population (n = 6 per group). (b, c) BODIPY 493/503 expression in IM (b) and AM (c) were analysed by flow cytometry at specified time points. Numbers in the plots indicate cell frequencies of BODIPY 493/503 positive macrophages. The scatter plots with bar graphs depict the MFI of BODIPY 493/503 in macrophages, normalised to the naïve SPD-fed group for each respective week (n = 6 per group). (d) RNA in situ hybridisation was performed to target Cd36 (pink), Cd68 (green), and mycobacterial 16S rRNA gene (red), coupled with DAPI staining (blue) to visualise nuclei, in lung tissue of LPD-fed mice at 10 weeks p.i. Representative images from five mice are displayed (400× magnification; scale bars = 20 μm). (e) Detection of M. avium acid-fast bacilli in lung tissue at 10 weeks p.i. using Ziehl-Neelsen staining, visualised with brightfield microscopy. Representative images from five mice are displayed (1000× magnification; scale bars = 10 μm). The results are presented as the mean ± SD, and statistical significance was determined by unpaired Student's t-test and one-way ANOVA with Tukey's multiple comparison test (NS, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001). SPD, standard protein diet; LPD, low-protein diet; p.i., post-infection; MFI, mean fluorescence intensity; FMO, fluorescence-minus-one; AM, alveolar macrophages; IM, interstitial macrophages.

Journal: eBioMedicine

Article Title: Protein-energy restriction-induced lipid metabolism disruption causes stable-to-progressive disease shift in Mycobacterium avium -infected female mice

doi: 10.1016/j.ebiom.2024.105198

Figure Lengend Snippet: Enhanced CD36 expression and neutral lipid accumulation in macrophages by protein-energy restriction during M. avium infection . (a) CD36 expression in immune cells in the lung analysed by flow cytometry at 10 weeks p.i. The scatter plots with bar graphs depict the MFI of CD36 expression. Fluorescence minus one control was used to define the negative population (n = 6 per group). (b, c) BODIPY 493/503 expression in IM (b) and AM (c) were analysed by flow cytometry at specified time points. Numbers in the plots indicate cell frequencies of BODIPY 493/503 positive macrophages. The scatter plots with bar graphs depict the MFI of BODIPY 493/503 in macrophages, normalised to the naïve SPD-fed group for each respective week (n = 6 per group). (d) RNA in situ hybridisation was performed to target Cd36 (pink), Cd68 (green), and mycobacterial 16S rRNA gene (red), coupled with DAPI staining (blue) to visualise nuclei, in lung tissue of LPD-fed mice at 10 weeks p.i. Representative images from five mice are displayed (400× magnification; scale bars = 20 μm). (e) Detection of M. avium acid-fast bacilli in lung tissue at 10 weeks p.i. using Ziehl-Neelsen staining, visualised with brightfield microscopy. Representative images from five mice are displayed (1000× magnification; scale bars = 10 μm). The results are presented as the mean ± SD, and statistical significance was determined by unpaired Student's t-test and one-way ANOVA with Tukey's multiple comparison test (NS, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001). SPD, standard protein diet; LPD, low-protein diet; p.i., post-infection; MFI, mean fluorescence intensity; FMO, fluorescence-minus-one; AM, alveolar macrophages; IM, interstitial macrophages.

Techniques: Expressing, Infection, Flow Cytometry, Fluorescence, Control, In Situ, Hybridization, Staining, Microscopy, Comparison

Enhancement of M. avium bacterial growth by CD36-dependent palmitic acid uptake in BMDMs under nutrient-limited conditions . (a) MFI of CD36 expression in BMDMs infected or uninfected with M. avium were analysed by flow cytometry at 72 h p.i. under specified nutrient conditions. (b) Quantification of intracellular M. avium load in BMDMs at 72 h p.i., with palmitic acid added at specified concentrations at 4 h p.i. (c) BODIPY 493/503 expression in BMDMs infected or uninfected with M. avium was analysed by flow cytometry at 72 h p.i. following treatment with palmitic acid (50 μM) at 4 h p.i. Numbers in the plots indicate frequencies of BODIPY 493/503-positive macrophages. Fluorescence minus one control was used to define the negative population. Bar graphs represent the relative MFI of BODIPY 493/503, normalised to uninfected cells. (d) Gene expression analysis in M. avium -infected BMDMs post 16 h p.i., following palmitic acid (50 μM) treatment at 4 h p.i. mRNA levels were quantified relative to β-actin and normalised to the non-palmitic acid-treated group. (e) Measurement of BODIPY FL C16 uptake in BMDMs pre-treated with SSO (200 μM) or αCD36 (10 μg/mL) for 4 h, followed by a 1 h incubation with 1 μg/mL BODIPY FL C16. (f) BMDMs were treated with SSO (200 μΜ) or αCD36 (10 μg/mL), followed by M. avium SMC #7-RFP infection after a 4 h interval. Palmitic acid (50 μM) was added at 4 h p.i., with confocal microscopy conducted at 72 h p.i. to measure BODIPY 493/503 (green). Bar graphs show relative green fluorescence intensity, normalised to the control group mean. Representative images from three independent experiments are shown (400× magnification; scale bars = 10 μm). (g) Quantification of intracellular bacterial load in M. avium -infected BMDMs treated with SSO or αCD36 at specified concentrations. BMDMs were treated with SSO or αCD36, followed by M. avium infection after a 4 h interval. Palmitic acid (50 μM) was added at 4 h p.i., and measurements taken at 72 h p.i. All experiments were conducted three times, and representative data are shown. The results are presented as the mean ± SD, and statistical significance was determined by unpaired Student's t-test and one-way ANOVA with Tukey's multiple comparison test (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). MFI, mean fluorescence intensity; BMDMs, bone marrow-derived macrophage; p.i., post-infection; FBS, fetal bovine serum; FMO, fluorescence-minus-one; CFU, colony-forming units; M. av , Mycobacterium avium ; Acsl3 , acyl-CoA synthetase long-chain family member 3; Dgat1 , diacylglycerol O-acyltransferase 1; Dgat2 , diacylglycerol O-acyltransferase 2; Atgl , adipose triglyceride lipase; SSO, sulfo-N-succinimidyl oleate; αCD36, anti-CD36 antibody.

Journal: eBioMedicine

Article Title: Protein-energy restriction-induced lipid metabolism disruption causes stable-to-progressive disease shift in Mycobacterium avium -infected female mice

doi: 10.1016/j.ebiom.2024.105198

Figure Lengend Snippet: Enhancement of M. avium bacterial growth by CD36-dependent palmitic acid uptake in BMDMs under nutrient-limited conditions . (a) MFI of CD36 expression in BMDMs infected or uninfected with M. avium were analysed by flow cytometry at 72 h p.i. under specified nutrient conditions. (b) Quantification of intracellular M. avium load in BMDMs at 72 h p.i., with palmitic acid added at specified concentrations at 4 h p.i. (c) BODIPY 493/503 expression in BMDMs infected or uninfected with M. avium was analysed by flow cytometry at 72 h p.i. following treatment with palmitic acid (50 μM) at 4 h p.i. Numbers in the plots indicate frequencies of BODIPY 493/503-positive macrophages. Fluorescence minus one control was used to define the negative population. Bar graphs represent the relative MFI of BODIPY 493/503, normalised to uninfected cells. (d) Gene expression analysis in M. avium -infected BMDMs post 16 h p.i., following palmitic acid (50 μM) treatment at 4 h p.i. mRNA levels were quantified relative to β-actin and normalised to the non-palmitic acid-treated group. (e) Measurement of BODIPY FL C16 uptake in BMDMs pre-treated with SSO (200 μM) or αCD36 (10 μg/mL) for 4 h, followed by a 1 h incubation with 1 μg/mL BODIPY FL C16. (f) BMDMs were treated with SSO (200 μΜ) or αCD36 (10 μg/mL), followed by M. avium SMC #7-RFP infection after a 4 h interval. Palmitic acid (50 μM) was added at 4 h p.i., with confocal microscopy conducted at 72 h p.i. to measure BODIPY 493/503 (green). Bar graphs show relative green fluorescence intensity, normalised to the control group mean. Representative images from three independent experiments are shown (400× magnification; scale bars = 10 μm). (g) Quantification of intracellular bacterial load in M. avium -infected BMDMs treated with SSO or αCD36 at specified concentrations. BMDMs were treated with SSO or αCD36, followed by M. avium infection after a 4 h interval. Palmitic acid (50 μM) was added at 4 h p.i., and measurements taken at 72 h p.i. All experiments were conducted three times, and representative data are shown. The results are presented as the mean ± SD, and statistical significance was determined by unpaired Student's t-test and one-way ANOVA with Tukey's multiple comparison test (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). MFI, mean fluorescence intensity; BMDMs, bone marrow-derived macrophage; p.i., post-infection; FBS, fetal bovine serum; FMO, fluorescence-minus-one; CFU, colony-forming units; M. av , Mycobacterium avium ; Acsl3 , acyl-CoA synthetase long-chain family member 3; Dgat1 , diacylglycerol O-acyltransferase 1; Dgat2 , diacylglycerol O-acyltransferase 2; Atgl , adipose triglyceride lipase; SSO, sulfo-N-succinimidyl oleate; αCD36, anti-CD36 antibody.

Techniques: Expressing, Infection, Flow Cytometry, Fluorescence, Control, Gene Expression, Incubation, Confocal Microscopy, Comparison, Derivative Assay

Blocking CD36 attenuates MAC-PD progression in mice under protein-energy restriction . (a) Schematic design of the in vivo experiment for M. avium aerosol infection. Mice fed with LPD were infected with M. avium . A monoclonal anti-CD36 antibody (αCD36; 200 μg per mouse) was administered two times per week via i.p. injection starting at 5 weeks p.i. (n = 5 per group). (b) RNA in situ hybridisation was performed to target the mycobacterial 16S rRNA gene (red), coupled with DAPI staining (blue) to visualise nuclei, in lung tissue at 10 weeks p.i. Representative images from five mice per group are displayed (400× magnification; scale bars = 20 μm). (c) Enumeration of lung bacterial CFU at 10 weeks p.i. for each mouse group (n = 5 per group). CFU counts were normalised to lung tissue weight. (d) The superior lobes of the right lung were examined through H&E staining in subsets of mice. Representative images from five mice per group at 10 weeks p.i. are displayed (400X: scale bars = 50 μm). The scatter plots with bar graphs depict the quantitative analysis of foam cells per microscopic field in lung tissues (e, f) Quantification of total FFA (e) and TAG (f) levels in lung tissue for each mouse group (n = 5 per group). The results are presented as the mean ± SD, and Mann–Whitney rank tests were used for group comparisons. (∗∗ p < 0.01). (g, h) BODIPY 493/503 expression in IM (g) and AM (h) were analysed by flow cytometry at 10 weeks p.i. Numbers in the plots indicate cell frequencies of BODIPY 493/503 positive macrophages. The scatter plots with bar graphs depict the MFI of BODIPY 493/503 in macrophages, normalised to the SPD-fed infected group (n = 5 per group). The results are presented as the mean ± SD, and statistical significance was determined by unpaired Student's t-test and one-way ANOVA with Tukey's multiple comparison tests (∗∗ p < 0.01; ∗∗∗ p < 0.001). SPD, standard protein diet; LPD, low protein diet; p.i., post-infection; CFU, colony-forming units; i.p., intraperitoneal; FFA, free fatty acid; TAG, triacylglycerol; MFI, mean fluorescence intensity.

Journal: eBioMedicine

Article Title: Protein-energy restriction-induced lipid metabolism disruption causes stable-to-progressive disease shift in Mycobacterium avium -infected female mice

doi: 10.1016/j.ebiom.2024.105198

Figure Lengend Snippet: Blocking CD36 attenuates MAC-PD progression in mice under protein-energy restriction . (a) Schematic design of the in vivo experiment for M. avium aerosol infection. Mice fed with LPD were infected with M. avium . A monoclonal anti-CD36 antibody (αCD36; 200 μg per mouse) was administered two times per week via i.p. injection starting at 5 weeks p.i. (n = 5 per group). (b) RNA in situ hybridisation was performed to target the mycobacterial 16S rRNA gene (red), coupled with DAPI staining (blue) to visualise nuclei, in lung tissue at 10 weeks p.i. Representative images from five mice per group are displayed (400× magnification; scale bars = 20 μm). (c) Enumeration of lung bacterial CFU at 10 weeks p.i. for each mouse group (n = 5 per group). CFU counts were normalised to lung tissue weight. (d) The superior lobes of the right lung were examined through H&E staining in subsets of mice. Representative images from five mice per group at 10 weeks p.i. are displayed (400X: scale bars = 50 μm). The scatter plots with bar graphs depict the quantitative analysis of foam cells per microscopic field in lung tissues (e, f) Quantification of total FFA (e) and TAG (f) levels in lung tissue for each mouse group (n = 5 per group). The results are presented as the mean ± SD, and Mann–Whitney rank tests were used for group comparisons. (∗∗ p < 0.01). (g, h) BODIPY 493/503 expression in IM (g) and AM (h) were analysed by flow cytometry at 10 weeks p.i. Numbers in the plots indicate cell frequencies of BODIPY 493/503 positive macrophages. The scatter plots with bar graphs depict the MFI of BODIPY 493/503 in macrophages, normalised to the SPD-fed infected group (n = 5 per group). The results are presented as the mean ± SD, and statistical significance was determined by unpaired Student's t-test and one-way ANOVA with Tukey's multiple comparison tests (∗∗ p < 0.01; ∗∗∗ p < 0.001). SPD, standard protein diet; LPD, low protein diet; p.i., post-infection; CFU, colony-forming units; i.p., intraperitoneal; FFA, free fatty acid; TAG, triacylglycerol; MFI, mean fluorescence intensity.

Techniques: Blocking Assay, In Vivo, Aerosol, Infection, Injection, In Situ, Hybridization, Staining, MANN-WHITNEY, Expressing, Flow Cytometry, Comparison, Fluorescence

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