fluorogenic ne substrate  (Santa Cruz Biotechnology)

Journal: Cell Death & Disease

Article Title: Neutrophil extracellular traps characterize caseating granulomas

doi: 10.1038/s41419-024-06892-3

Figure Lengend Snippet: We detected NETs by the quantification of circulating NET degradation products and found that they are elevated in patients with TB when compared to NHD. a We quantified cfDNA in the sera by PicoGreen fluorescence, b MPO–DNA and c NE-DNA complexes by ELISA and d NE activity by the conversion of a specific fluorogenic substrate. e Summarizes mean values and ranges for all parameters. f – m Clinical parameters were associated with the results obtained by NET formation analyses of TB sera. f NE-DNA complexes, g MPO–DNA complexes, h NE activity and i neutrophil counts in the sera of TB patients, associated with the type of TB case: new (green) versus relapse (red). Note that NET formation is increased in the sera of TB patients in relapse. j NE-DNA complexes, k MPO–DNA complexes, l NE activity and m neutrophil counts in the sera of TB patients, grouped by the extent of tissue destruction: no destruction (green) or destruction (red). Note that NET formation, NE activity and neutrophil counts are increased in patients with tissue destruction. All statistical analyses were performed employing the Mann–Whitney test. Data are presented as mean ± standard deviation (s.d.). At least three technical replicates were obtained for each data set. NHD normal healthy donors, TB tuberculosis, MPO myeloperoxidase, NE neutrophil elastase, MFI mean fluorescence intensity, n.s. not significant.

Article Snippet: To assess the activity of NE in the sera of patients with TB, 10 µL of serum and 100 µM fluorogenic NE substrate (MeOSuc-AAPV-AMC, sc-201163, Santa Cruz Biotechnology Inc., Dallas, TX, USA) per well were diluted in Dulbecco´s phosphate buffered saline (DPBS, Thermo Fisher Scientific Inc., Waltham, MA, USA) in a 384-well plate (#3680, Corning Inc., Corning, NY, USA).

Techniques: Fluorescence, Enzyme-linked Immunosorbent Assay, Activity Assay, MANN-WHITNEY, Standard Deviation

Journal: JCI Insight

Article Title: Neutrophil proteases are protective against SARS-CoV-2 by degrading the spike protein and dampening virus-mediated inflammation

doi: 10.1172/jci.insight.174133

Figure Lengend Snippet: ( A and B ) SDS-PAGE of recombinant trimeric S(614G) ( A ) and S(MA10) ( B ) incubated with purified human CatG, NE, and PR3 at indicated concentrations. Arrowheads indicate S protein and asterisks cleaved fragments. ( C and D ) Immunoblots of recombinant trimeric S(614G) and S(MA10) incubated with mouse neutrophil lysates with or without α1AT and probed with anti-S1 ( C ) or anti-S2 ( D ) subunit antibodies. ( E ) Titers of VSV*ΔG-S Δ21 incubated with NSPs. ( F and G ) Titers of SARS-CoV-2 614G ( F ) and SARS-CoV-2 MA10 ( G ) incubated with NSPs. Data in A – D representative of at least 3 independent experiments. Data in E – G were analyzed by 1-way ANOVA with Dunnett’s multiple-comparison test, comparing the NSP-treated group to the PBS control ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The proteolytic activity of the human purified NSPs was determined with chromogenic peptide substrates specific for CatG (Ala-Ala-Pro-Phe-pNA) (Sigma-Aldrich) and NE or PR3 (Ala-Ala-Pro-Val-pNA) (Calbiochem).

Techniques: SDS Page, Recombinant, Incubation, Purification, Western Blot, Comparison, Control

Journal: JCI Insight

Article Title: Neutrophil proteases are protective against SARS-CoV-2 by degrading the spike protein and dampening virus-mediated inflammation

doi: 10.1172/jci.insight.174133

Figure Lengend Snippet: WT, CatG –/– , NE –/– , PR3 –/– , and NE.CatG –/– mice were inoculated intranasally with 1 × 10 4 TCID 50 SARS-CoV-2 MA10 . ( A ) Relative body weight change overtime after infection. Data were analyzed by 2-way ANOVA with Dunett’s multiple-comparison test, comparing the knockout groups to the WT group. Data were pooled from 6 independent experiments ( n = 12–31 mice/group). * P < 0.05; color of asterisk corresponds to the genotype. ( B ) Viral copy numbers were determined by RT-qPCR in oropharyngeal swabs or tissue homogenates. ( C ) Viral titers in lung homogenates. ( B and C ) Data were log 10 transformed and analyzed by 1-way ANOVA with Dunnett’s multiple-comparison test comparing the knockout groups to the WT group at each time point. Data pooled from 7 independent experiments (2 dpi, n = 16–19/group; 4 dpi, n = 10–17/group). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The proteolytic activity of the human purified NSPs was determined with chromogenic peptide substrates specific for CatG (Ala-Ala-Pro-Phe-pNA) (Sigma-Aldrich) and NE or PR3 (Ala-Ala-Pro-Val-pNA) (Calbiochem).

Techniques: Infection, Comparison, Knock-Out, Quantitative RT-PCR, Transformation Assay

Journal: JCI Insight

Article Title: Neutrophil proteases are protective against SARS-CoV-2 by degrading the spike protein and dampening virus-mediated inflammation

doi: 10.1172/jci.insight.174133

Figure Lengend Snippet: ( A and B ) WT, CatG –/– , NE –/– , PR3 –/– , and NE.CatG –/– mice were inoculated intranasally with 1 × 10 4 TCID 50 SARS-CoV-2 MA10 . Cytokines ( A ) and chemokines ( B ) were measured in lung homogenates on the indicated dpi. Data were analyzed by 1-way ANOVA with Dunnett’s multiple-comparison test comparing the groups of knockout mice to the WT group at each time point. Data pooled from 7 independent experiments (2 dpi, n = 8–11/group; 4dpi, n = 10–19/group). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The proteolytic activity of the human purified NSPs was determined with chromogenic peptide substrates specific for CatG (Ala-Ala-Pro-Phe-pNA) (Sigma-Aldrich) and NE or PR3 (Ala-Ala-Pro-Val-pNA) (Calbiochem).

Techniques: Comparison, Knock-Out