ne inhibitor nei Search Results


neutrophil elastase ne inhibitor nei  (MedChemExpress)
94
MedChemExpress neutrophil elastase ne inhibitor nei
Fig. 1. PVL induces ROS-independent NET formation. (A, B) ROS formation was quantified in control (A: n = 14, B: n = 3) or CGD human (n = 3) primary <t>neutrophils</t> stimulated with PMA (50 nM) or PVL (0.1 nM, 1 nM or 10 nM). (C) Control or CGD neutrophils were stimulated with PMA (50 nM) or PVL (10 nM) for 3 h, and cell death was quantified using the cell-impermeable DNA dye SYTOX Green. Indicated is SYTOX Green fluorescence relative to t = 0 min. (D) Representative confocal microscopy images of control and CGD patient neutrophils either naïve or stimulated with PMA (50 nM) or PVL (1 nM or 10 nM) and stained for DNA (blue), NE (red), and chromatin (green). The scale bar represents 10 µm. (E–G) Healthy human neutrophils were treated with PKC inhibitor Gö6983 (1 μM), BAPTA-AM (10 μM), DPI (1 μM), pyrocatechol (30 μM), ABAH (500 μM), NEi (20 μM), and AEBSF (100 μM) for 30 min, before stimulating with (E) PVL 1 nM, (F) PVL 10 nM, or (G) PMA 50 nM for 4 h. We quantified cell death with SYTOX Green and the fluorescence signal relative to naïve is indicated. (H) Representative confocal microscopy of naïve neutrophils or after stimulation with PMA or PVL (1 nM or 10 nM) in the presence or absence of indicated inhibitors, and stained for DNA (blue), NE (red), and chromatin (green). The scale bar represents 10 µm. (C) Two-way analysis of variance with Tukey’s post hoc test. *P < 0.05, ***P < 0.001. (E–G) Mean ± SD of 4 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 1-way analysis of variance with Dunnett’s multiple comparison test. ns = not significant.
Neutrophil Elastase Ne Inhibitor Nei, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 1 article reviews
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o -methoxy-succinyl-alanyl-alanyl-prolyl-alanyl-chloro-methylketone  (Millipore)
90
Millipore o -methoxy-succinyl-alanyl-alanyl-prolyl-alanyl-chloro-methylketone
A , B ) NE is required for the survival and intracellular growth of L. donovani in macrophages. Peritoneal thioglycolate-recruited macrophages from C57BL/6 mice ( A ) or from NE knockout mice ( ela 2 −/− ) ( B ) were cultivated on glass coverslips overnight in RPMI-FCS and washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI supplemented with 0.1% BSA. C ) The monolayers were washed 3 times to remove extracellular parasites, fixed with methanol and Giemsa stained (3 h), or further incubated for 24 h ( A , B ), 48 h, or 72 h in RPMI-FCS at 37°C before fixation and staining. The number of intracellular parasites was determined under the light microscope. Where indicated, <t>the</t> <t>irreversible</t> <t>NEI</t> OMeSuc-Ala-Ala-Pro-Val-CMK was added to the cultures at 10 μM final concentration immediately before addition of the parasites and kept for the 3 h infection. D ) Macrophages were infected as previously described for 3 h, and the monolayers were washed for removal of extracellular parasites and fixed (3 h) or cultured in RPMI-FCS for 72 h before fixation and staining. Where indicated (+), active purified human NE was added at 200 ng/ml final concentration to the cultures after the 3 h infection and remained for the 72 h. The experiments were performed in triplicate and repeated at least 3 independent times. The graphs show 1 representative experiment in triplicate. The graphs show means ± sd ; statistical significance was assessed using 1-way ANOVA and the Bonferroni posttest. * P < 0.05, ** P < 0.01, **** P < 0.0001.
O Methoxy Succinyl Alanyl Alanyl Prolyl Alanyl Chloro Methylketone, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/o -methoxy-succinyl-alanyl-alanyl-prolyl-alanyl-chloro-methylketone/product/Millipore
Average 90 stars, based on 1 article reviews
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irreversible ne inhibitor iii (nei  (Millipore)
90
Millipore irreversible ne inhibitor iii (nei
A , B ) NE is required for the survival and intracellular growth of L. donovani in macrophages. Peritoneal thioglycolate-recruited macrophages from C57BL/6 mice ( A ) or from NE knockout mice ( ela 2 −/− ) ( B ) were cultivated on glass coverslips overnight in RPMI-FCS and washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI supplemented with 0.1% BSA. C ) The monolayers were washed 3 times to remove extracellular parasites, fixed with methanol and Giemsa stained (3 h), or further incubated for 24 h ( A , B ), 48 h, or 72 h in RPMI-FCS at 37°C before fixation and staining. The number of intracellular parasites was determined under the light microscope. Where indicated, <t>the</t> <t>irreversible</t> <t>NEI</t> OMeSuc-Ala-Ala-Pro-Val-CMK was added to the cultures at 10 μM final concentration immediately before addition of the parasites and kept for the 3 h infection. D ) Macrophages were infected as previously described for 3 h, and the monolayers were washed for removal of extracellular parasites and fixed (3 h) or cultured in RPMI-FCS for 72 h before fixation and staining. Where indicated (+), active purified human NE was added at 200 ng/ml final concentration to the cultures after the 3 h infection and remained for the 72 h. The experiments were performed in triplicate and repeated at least 3 independent times. The graphs show 1 representative experiment in triplicate. The graphs show means ± sd ; statistical significance was assessed using 1-way ANOVA and the Bonferroni posttest. * P < 0.05, ** P < 0.01, **** P < 0.0001.
Irreversible Ne Inhibitor Iii (Nei, supplied by Millipore, 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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Average 90 stars, based on 1 article reviews
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o -methoxy-succinyl-alanyl-alanyl-prolyl-alanyl-chloromethylketone (omesuc-aapv-cmk)  (Millipore)
90
Millipore o -methoxy-succinyl-alanyl-alanyl-prolyl-alanyl-chloromethylketone (omesuc-aapv-cmk)
A , B ) NE is required for the survival and intracellular growth of L. donovani in macrophages. Peritoneal thioglycolate-recruited macrophages from C57BL/6 mice ( A ) or from NE knockout mice ( ela 2 −/− ) ( B ) were cultivated on glass coverslips overnight in RPMI-FCS and washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI supplemented with 0.1% BSA. C ) The monolayers were washed 3 times to remove extracellular parasites, fixed with methanol and Giemsa stained (3 h), or further incubated for 24 h ( A , B ), 48 h, or 72 h in RPMI-FCS at 37°C before fixation and staining. The number of intracellular parasites was determined under the light microscope. Where indicated, <t>the</t> <t>irreversible</t> <t>NEI</t> OMeSuc-Ala-Ala-Pro-Val-CMK was added to the cultures at 10 μM final concentration immediately before addition of the parasites and kept for the 3 h infection. D ) Macrophages were infected as previously described for 3 h, and the monolayers were washed for removal of extracellular parasites and fixed (3 h) or cultured in RPMI-FCS for 72 h before fixation and staining. Where indicated (+), active purified human NE was added at 200 ng/ml final concentration to the cultures after the 3 h infection and remained for the 72 h. The experiments were performed in triplicate and repeated at least 3 independent times. The graphs show 1 representative experiment in triplicate. The graphs show means ± sd ; statistical significance was assessed using 1-way ANOVA and the Bonferroni posttest. * P < 0.05, ** P < 0.01, **** P < 0.0001.
O Methoxy Succinyl Alanyl Alanyl Prolyl Alanyl Chloromethylketone (Omesuc Aapv Cmk), supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/o -methoxy-succinyl-alanyl-alanyl-prolyl-alanyl-chloromethylketone (omesuc-aapv-cmk)/product/Millipore
Average 90 stars, based on 1 article reviews
o -methoxy-succinyl-alanyl-alanyl-prolyl-alanyl-chloromethylketone (omesuc-aapv-cmk) - by Bioz Stars, 2026-03
90/100 stars
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ne inhibitor nei  (MedChemExpress)
95
MedChemExpress ne inhibitor nei
A , B ) NE is required for the survival and intracellular growth of L. donovani in macrophages. Peritoneal thioglycolate-recruited macrophages from C57BL/6 mice ( A ) or from NE knockout mice ( ela 2 −/− ) ( B ) were cultivated on glass coverslips overnight in RPMI-FCS and washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI supplemented with 0.1% BSA. C ) The monolayers were washed 3 times to remove extracellular parasites, fixed with methanol and Giemsa stained (3 h), or further incubated for 24 h ( A , B ), 48 h, or 72 h in RPMI-FCS at 37°C before fixation and staining. The number of intracellular parasites was determined under the light microscope. Where indicated, <t>the</t> <t>irreversible</t> <t>NEI</t> OMeSuc-Ala-Ala-Pro-Val-CMK was added to the cultures at 10 μM final concentration immediately before addition of the parasites and kept for the 3 h infection. D ) Macrophages were infected as previously described for 3 h, and the monolayers were washed for removal of extracellular parasites and fixed (3 h) or cultured in RPMI-FCS for 72 h before fixation and staining. Where indicated (+), active purified human NE was added at 200 ng/ml final concentration to the cultures after the 3 h infection and remained for the 72 h. The experiments were performed in triplicate and repeated at least 3 independent times. The graphs show 1 representative experiment in triplicate. The graphs show means ± sd ; statistical significance was assessed using 1-way ANOVA and the Bonferroni posttest. * P < 0.05, ** P < 0.01, **** P < 0.0001.
Ne Inhibitor Nei, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ne inhibitor nei/product/MedChemExpress
Average 95 stars, based on 1 article reviews
ne inhibitor nei - by Bioz Stars, 2026-03
95/100 stars
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heneicosanoic acid  (TargetMol)
N/A
Heneicosanoic acid HEA is a fatty acid found in human milk fat HEA is also a part of the phospholipids of the articular cartilage boundary lubricant HEA is a constituent of red blood cell fatty
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m dimethylaminomethyleneiminophenol hydrochloride  (TargetMol)
N/A
m Dimethylaminomethyleneiminophenol hydrochloride is a bioactive chemical
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methyl heneicosanoate  (TargetMol)
N/A
Methyl heneicosanoate is a natural product
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isoboonein acetate  (TargetMol)
N/A
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fortuneine  (TargetMol)
N/A
Fortuneine is a natural product from Cephalotaxus fortunei
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l ergothioneine  (TargetMol)
N/A
Ergothioneine is synthesized by certain bacteria and fungi It is generally considered an antioxidant
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m dimethylaminomethyleneiminophenol  (TargetMol)
N/A
m Dimethylaminomethyleneiminophenol is a bioactive chemical
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Image Search Results


Fig. 1. PVL induces ROS-independent NET formation. (A, B) ROS formation was quantified in control (A: n = 14, B: n = 3) or CGD human (n = 3) primary neutrophils stimulated with PMA (50 nM) or PVL (0.1 nM, 1 nM or 10 nM). (C) Control or CGD neutrophils were stimulated with PMA (50 nM) or PVL (10 nM) for 3 h, and cell death was quantified using the cell-impermeable DNA dye SYTOX Green. Indicated is SYTOX Green fluorescence relative to t = 0 min. (D) Representative confocal microscopy images of control and CGD patient neutrophils either naïve or stimulated with PMA (50 nM) or PVL (1 nM or 10 nM) and stained for DNA (blue), NE (red), and chromatin (green). The scale bar represents 10 µm. (E–G) Healthy human neutrophils were treated with PKC inhibitor Gö6983 (1 μM), BAPTA-AM (10 μM), DPI (1 μM), pyrocatechol (30 μM), ABAH (500 μM), NEi (20 μM), and AEBSF (100 μM) for 30 min, before stimulating with (E) PVL 1 nM, (F) PVL 10 nM, or (G) PMA 50 nM for 4 h. We quantified cell death with SYTOX Green and the fluorescence signal relative to naïve is indicated. (H) Representative confocal microscopy of naïve neutrophils or after stimulation with PMA or PVL (1 nM or 10 nM) in the presence or absence of indicated inhibitors, and stained for DNA (blue), NE (red), and chromatin (green). The scale bar represents 10 µm. (C) Two-way analysis of variance with Tukey’s post hoc test. *P < 0.05, ***P < 0.001. (E–G) Mean ± SD of 4 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 1-way analysis of variance with Dunnett’s multiple comparison test. ns = not significant.

Journal: Journal of leukocyte biology

Article Title: Panton-Valentine leukocidin-induced neutrophil extracellular traps lack antimicrobial activity and are readily induced in patients with recurrent PVL+-Staphylococcus aureus infections.

doi: 10.1093/jleuko/qiad137

Figure Lengend Snippet: Fig. 1. PVL induces ROS-independent NET formation. (A, B) ROS formation was quantified in control (A: n = 14, B: n = 3) or CGD human (n = 3) primary neutrophils stimulated with PMA (50 nM) or PVL (0.1 nM, 1 nM or 10 nM). (C) Control or CGD neutrophils were stimulated with PMA (50 nM) or PVL (10 nM) for 3 h, and cell death was quantified using the cell-impermeable DNA dye SYTOX Green. Indicated is SYTOX Green fluorescence relative to t = 0 min. (D) Representative confocal microscopy images of control and CGD patient neutrophils either naïve or stimulated with PMA (50 nM) or PVL (1 nM or 10 nM) and stained for DNA (blue), NE (red), and chromatin (green). The scale bar represents 10 µm. (E–G) Healthy human neutrophils were treated with PKC inhibitor Gö6983 (1 μM), BAPTA-AM (10 μM), DPI (1 μM), pyrocatechol (30 μM), ABAH (500 μM), NEi (20 μM), and AEBSF (100 μM) for 30 min, before stimulating with (E) PVL 1 nM, (F) PVL 10 nM, or (G) PMA 50 nM for 4 h. We quantified cell death with SYTOX Green and the fluorescence signal relative to naïve is indicated. (H) Representative confocal microscopy of naïve neutrophils or after stimulation with PMA or PVL (1 nM or 10 nM) in the presence or absence of indicated inhibitors, and stained for DNA (blue), NE (red), and chromatin (green). The scale bar represents 10 µm. (C) Two-way analysis of variance with Tukey’s post hoc test. *P < 0.05, ***P < 0.001. (E–G) Mean ± SD of 4 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 1-way analysis of variance with Dunnett’s multiple comparison test. ns = not significant.

Article Snippet: We used the following inhibitors: Gö6983 (protein kinase C [PKC] inhibitor; Tocris) BAPTA-AM (Thermo Fisher Scientific), pyrocatechol (Sigma-Aldrich), ABAH (Cayman chemical), neutrophil elastase (NE) inhibitor (NEi) (MedChemExpress), DPI (Calbiochem), AEBSF (Sigma-Aldrich), allopurinol (Sigma-Aldrich), Apamin (Sigma-Aldrich), DNP (Sigma-Aldrich) and FCCP (Abcam).

Techniques: Control, Fluorescence, Confocal Microscopy, Staining, Comparison

Fig. 2. NET proteome assembly is different in PVL-induced NETs. (A) Principal component analysis of the proteomes of naïve neutrophils, and PVL-induced (10 nM), PMA-induced (50 nM), and nigericin-induced (15 µM) NETs. Analysis was performed on scaled log2-abundance of all proteins detected in at least 2 of 3 replicates. (B) Euler diagram showing the number and distribution of significant differentially abundant peptides (logFC > 1, adjusted P < 0.01) on NETs compared with naïve neutrophils. Areas are proportional to DAP set size. (C) Clustered heatmap showing fold change (log2) values on each NET sample compared with naïve neutrophils of significant DAPs from panel B. Clustering was performed by k-means algorithm with k = 4 clusters. (D, E) Top 25 most abundant DAPs that are significantly differentially abundant on PVL-induced NETs compared with PMA-induced (D) and nigericin-induced NETs (E). Point size and fill color represent average abundance across samples and adjusted P value, respectively. Proteomes were made from n = 3 samples per condition from independent donors. DAP significance for each comparison was determined by a threshold of |[log2 fold change]| > 1 and adjusted P value < 0.01.

Journal: Journal of leukocyte biology

Article Title: Panton-Valentine leukocidin-induced neutrophil extracellular traps lack antimicrobial activity and are readily induced in patients with recurrent PVL+-Staphylococcus aureus infections.

doi: 10.1093/jleuko/qiad137

Figure Lengend Snippet: Fig. 2. NET proteome assembly is different in PVL-induced NETs. (A) Principal component analysis of the proteomes of naïve neutrophils, and PVL-induced (10 nM), PMA-induced (50 nM), and nigericin-induced (15 µM) NETs. Analysis was performed on scaled log2-abundance of all proteins detected in at least 2 of 3 replicates. (B) Euler diagram showing the number and distribution of significant differentially abundant peptides (logFC > 1, adjusted P < 0.01) on NETs compared with naïve neutrophils. Areas are proportional to DAP set size. (C) Clustered heatmap showing fold change (log2) values on each NET sample compared with naïve neutrophils of significant DAPs from panel B. Clustering was performed by k-means algorithm with k = 4 clusters. (D, E) Top 25 most abundant DAPs that are significantly differentially abundant on PVL-induced NETs compared with PMA-induced (D) and nigericin-induced NETs (E). Point size and fill color represent average abundance across samples and adjusted P value, respectively. Proteomes were made from n = 3 samples per condition from independent donors. DAP significance for each comparison was determined by a threshold of |[log2 fold change]| > 1 and adjusted P value < 0.01.

Article Snippet: We used the following inhibitors: Gö6983 (protein kinase C [PKC] inhibitor; Tocris) BAPTA-AM (Thermo Fisher Scientific), pyrocatechol (Sigma-Aldrich), ABAH (Cayman chemical), neutrophil elastase (NE) inhibitor (NEi) (MedChemExpress), DPI (Calbiochem), AEBSF (Sigma-Aldrich), allopurinol (Sigma-Aldrich), Apamin (Sigma-Aldrich), DNP (Sigma-Aldrich) and FCCP (Abcam).

Techniques: Comparison

Fig. 3. PVL-induced NETs do not kill MRSA. (A) Primary human neutrophils were stimulated with PVL (100 nM), PMA (100 nM), or nigericin (30 µM) for 4 h to induce NETs. MRSA was incubated with these NETs for 1 h and colony-forming units was quantified after overnight incubation on TSA plates. Bacterial viability is expressed as a percentage of colony-forming units normalized to MRSA incubated in the absence of NETs. (B) The DNA content of PVL-, PMA-, and nigericin-induced NETs at 4 h was quantified by spectrophotometry. (A) Mean ± SD of 5 independent experiments. *P < 0.05, *** P < 0.001, 1-way analysis of variance. (B) Mean ± SD of 4 independent experiments. ns = not significant.

Journal: Journal of leukocyte biology

Article Title: Panton-Valentine leukocidin-induced neutrophil extracellular traps lack antimicrobial activity and are readily induced in patients with recurrent PVL+-Staphylococcus aureus infections.

doi: 10.1093/jleuko/qiad137

Figure Lengend Snippet: Fig. 3. PVL-induced NETs do not kill MRSA. (A) Primary human neutrophils were stimulated with PVL (100 nM), PMA (100 nM), or nigericin (30 µM) for 4 h to induce NETs. MRSA was incubated with these NETs for 1 h and colony-forming units was quantified after overnight incubation on TSA plates. Bacterial viability is expressed as a percentage of colony-forming units normalized to MRSA incubated in the absence of NETs. (B) The DNA content of PVL-, PMA-, and nigericin-induced NETs at 4 h was quantified by spectrophotometry. (A) Mean ± SD of 5 independent experiments. *P < 0.05, *** P < 0.001, 1-way analysis of variance. (B) Mean ± SD of 4 independent experiments. ns = not significant.

Article Snippet: We used the following inhibitors: Gö6983 (protein kinase C [PKC] inhibitor; Tocris) BAPTA-AM (Thermo Fisher Scientific), pyrocatechol (Sigma-Aldrich), ABAH (Cayman chemical), neutrophil elastase (NE) inhibitor (NEi) (MedChemExpress), DPI (Calbiochem), AEBSF (Sigma-Aldrich), allopurinol (Sigma-Aldrich), Apamin (Sigma-Aldrich), DNP (Sigma-Aldrich) and FCCP (Abcam).

Techniques: Incubation, Spectrophotometry

Fig. 4. Neutrophils from patients with recurrent PVL-SA infections make more NETs in response to PVL and express more CD45 than neutrophils from healthy donors. (A) Purified primary neutrophils from patients experiencing recurrent PVL-SA infections (n = 19) and healthy control individuals (n = 19) were either left untreated or stimulated with PMA (50 nM) or PVL (0.1 nM, 1 nM, or 10 nM) for 4 h, and cell death was quantified by adding the cell-impermeable DNA dye SYTOX Green and measuring fluorescence. The boxplot indicates SYTOX fluorescence relative to naïve neutrophils at 4 h. (B) CD45, C5L2, CD88, CD63, and CD66b were immunolabelled on neutrophils from patients and healthy control individuals. The fluorescence was measured and indicated as log-transformed mean fluorescence intensity (MFI). (C) CD45 or (D) C5L2 fluorescence of patient neutrophils relative to control neutrophils was plotted against SYTOX fluorescence of patient neutrophils relative to control neutrophils after incubation with 0.1 nM PVL for 4 h. (A) Unpaired Wilcoxon signed rank test with Bonferroni post hoc test, *P < 0.05. (B) Paired t test, ****P < 0.0001. (C, D) Spearman’s correlation test in which the coefficient of correlation (ρ) and probability (P) are indicated. A best-fit line indicates the trend.

Journal: Journal of leukocyte biology

Article Title: Panton-Valentine leukocidin-induced neutrophil extracellular traps lack antimicrobial activity and are readily induced in patients with recurrent PVL+-Staphylococcus aureus infections.

doi: 10.1093/jleuko/qiad137

Figure Lengend Snippet: Fig. 4. Neutrophils from patients with recurrent PVL-SA infections make more NETs in response to PVL and express more CD45 than neutrophils from healthy donors. (A) Purified primary neutrophils from patients experiencing recurrent PVL-SA infections (n = 19) and healthy control individuals (n = 19) were either left untreated or stimulated with PMA (50 nM) or PVL (0.1 nM, 1 nM, or 10 nM) for 4 h, and cell death was quantified by adding the cell-impermeable DNA dye SYTOX Green and measuring fluorescence. The boxplot indicates SYTOX fluorescence relative to naïve neutrophils at 4 h. (B) CD45, C5L2, CD88, CD63, and CD66b were immunolabelled on neutrophils from patients and healthy control individuals. The fluorescence was measured and indicated as log-transformed mean fluorescence intensity (MFI). (C) CD45 or (D) C5L2 fluorescence of patient neutrophils relative to control neutrophils was plotted against SYTOX fluorescence of patient neutrophils relative to control neutrophils after incubation with 0.1 nM PVL for 4 h. (A) Unpaired Wilcoxon signed rank test with Bonferroni post hoc test, *P < 0.05. (B) Paired t test, ****P < 0.0001. (C, D) Spearman’s correlation test in which the coefficient of correlation (ρ) and probability (P) are indicated. A best-fit line indicates the trend.

Article Snippet: We used the following inhibitors: Gö6983 (protein kinase C [PKC] inhibitor; Tocris) BAPTA-AM (Thermo Fisher Scientific), pyrocatechol (Sigma-Aldrich), ABAH (Cayman chemical), neutrophil elastase (NE) inhibitor (NEi) (MedChemExpress), DPI (Calbiochem), AEBSF (Sigma-Aldrich), allopurinol (Sigma-Aldrich), Apamin (Sigma-Aldrich), DNP (Sigma-Aldrich) and FCCP (Abcam).

Techniques: Purification, Control, Fluorescence, Transformation Assay, Incubation

A , B ) NE is required for the survival and intracellular growth of L. donovani in macrophages. Peritoneal thioglycolate-recruited macrophages from C57BL/6 mice ( A ) or from NE knockout mice ( ela 2 −/− ) ( B ) were cultivated on glass coverslips overnight in RPMI-FCS and washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI supplemented with 0.1% BSA. C ) The monolayers were washed 3 times to remove extracellular parasites, fixed with methanol and Giemsa stained (3 h), or further incubated for 24 h ( A , B ), 48 h, or 72 h in RPMI-FCS at 37°C before fixation and staining. The number of intracellular parasites was determined under the light microscope. Where indicated, the irreversible NEI OMeSuc-Ala-Ala-Pro-Val-CMK was added to the cultures at 10 μM final concentration immediately before addition of the parasites and kept for the 3 h infection. D ) Macrophages were infected as previously described for 3 h, and the monolayers were washed for removal of extracellular parasites and fixed (3 h) or cultured in RPMI-FCS for 72 h before fixation and staining. Where indicated (+), active purified human NE was added at 200 ng/ml final concentration to the cultures after the 3 h infection and remained for the 72 h. The experiments were performed in triplicate and repeated at least 3 independent times. The graphs show 1 representative experiment in triplicate. The graphs show means ± sd ; statistical significance was assessed using 1-way ANOVA and the Bonferroni posttest. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Journal: The FASEB Journal

Article Title: Neutrophil elastase promotes Leishmania donovani infection via interferon-β

doi: 10.1096/fj.201900524R

Figure Lengend Snippet: A , B ) NE is required for the survival and intracellular growth of L. donovani in macrophages. Peritoneal thioglycolate-recruited macrophages from C57BL/6 mice ( A ) or from NE knockout mice ( ela 2 −/− ) ( B ) were cultivated on glass coverslips overnight in RPMI-FCS and washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI supplemented with 0.1% BSA. C ) The monolayers were washed 3 times to remove extracellular parasites, fixed with methanol and Giemsa stained (3 h), or further incubated for 24 h ( A , B ), 48 h, or 72 h in RPMI-FCS at 37°C before fixation and staining. The number of intracellular parasites was determined under the light microscope. Where indicated, the irreversible NEI OMeSuc-Ala-Ala-Pro-Val-CMK was added to the cultures at 10 μM final concentration immediately before addition of the parasites and kept for the 3 h infection. D ) Macrophages were infected as previously described for 3 h, and the monolayers were washed for removal of extracellular parasites and fixed (3 h) or cultured in RPMI-FCS for 72 h before fixation and staining. Where indicated (+), active purified human NE was added at 200 ng/ml final concentration to the cultures after the 3 h infection and remained for the 72 h. The experiments were performed in triplicate and repeated at least 3 independent times. The graphs show 1 representative experiment in triplicate. The graphs show means ± sd ; statistical significance was assessed using 1-way ANOVA and the Bonferroni posttest. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Where indicated in the figure legends, the irreversible inhibitor of NE (NEI), O -methoxy-succinyl-alanyl-alanyl-prolyl-alanyl-chloro-methylketone (10 μM; Calbiochem, San Diego, CA, USA), was added to the macrophages 5 min before addition of the parasites.

Techniques: Knock-Out, Infection, Staining, Incubation, Light Microscopy, Concentration Assay, Cell Culture, Purification