mouse anti-human ll37  (Absolute Biotech Inc)

Journal: Annals of Translational Medicine

Article Title: Telomere dysfunction promotes small vessel vasculitis via the LL37-NETs-dependent mechanism

doi: 10.21037/atm.2020.02.130

Figure Lengend Snippet: Higher levels of LL37 expression and NET release are determined in patients with SVV. (A) Telomere length of SVV and HCs by real-time Q-PCR; (B) levels of LL37 in serum from the disease group and healthy group; (C) levels of LL37 in urine from the disease group and healthy group; (D) levels of NET remnants in serum from patients with SVV or HCs; (E) levels of cf-DNA in serum from patients with SVV or HCs (A-E). N=70 for each group. The error bars of the graph represent the SEM. Unpaired Student’s t-test was conducted to analyze the statistical significance of two independent experiments; (F) an Alexa 488-labeled antibody against H2A-H2B-DNA complexes was used to visualize NETs in green, and DAPI was used to stain the nuclei in blue. Pictures were merged to form an overlay image; (G) H2A-H2B-DNA complexes, LL37, and nuclei are stained green, red, and blue, respectively. Pictures were merged to form an overlay image. A.U., arbitrary unit.

Article Snippet: A FITC-labeled PNA probe specific for (TTAGGG)n sequences (PANAGENE, Korea) was co-denatured with the slide at 80 °C for 3 min and hybridized at RT for 2 h. Slides were then incubated first with the mouse-anti-human LL37 (HM2070, Hycult biotech, The Netherlands) and rabbit-anti-γH2AX (MABE205, Millipore, USA) antibodies, followed by the secondary Alexa-Fluor-594-labeled goat-anti-mouse antibody and Alexa-Fluor-488-labeled goat-anti-rabbit antibody.

Techniques: Expressing, Labeling, Staining

Journal: Annals of Translational Medicine

Article Title: Telomere dysfunction promotes small vessel vasculitis via the LL37-NETs-dependent mechanism

doi: 10.21037/atm.2020.02.130

Figure Lengend Snippet: Immune aging of neutrophils and inflammatory medium release are determined in situ in individuals with SVV. (A) Neutrophils (polymorphic nuclear) infiltrated the kidney tissue from patients with SVV. Telomere dysfunction was found by the co-localization of γH2AX (purple) and telomeres (green) in the nucleus (DNA in blue). Immunostaining of LL37 showed perinuclear LL37 (red) in neutrophils that suffered DNA dysfunction; (B) H2A-H2B-DNA complexes, LL37, and nuclei are stained green, red, and blue, respectively. Pictures were merged to form an overlay image. LL37 assembled around the polymorphic nucleus in a background of NET deposition in the kidney tissue of SVV patients.

Article Snippet: A FITC-labeled PNA probe specific for (TTAGGG)n sequences (PANAGENE, Korea) was co-denatured with the slide at 80 °C for 3 min and hybridized at RT for 2 h. Slides were then incubated first with the mouse-anti-human LL37 (HM2070, Hycult biotech, The Netherlands) and rabbit-anti-γH2AX (MABE205, Millipore, USA) antibodies, followed by the secondary Alexa-Fluor-594-labeled goat-anti-mouse antibody and Alexa-Fluor-488-labeled goat-anti-rabbit antibody.

Techniques: In Situ, Immunostaining, Staining

Journal: Annals of Translational Medicine

Article Title: Telomere dysfunction promotes small vessel vasculitis via the LL37-NETs-dependent mechanism

doi: 10.21037/atm.2020.02.130

Figure Lengend Snippet: Human neutrophils from SVV produce more LL37-mediated-NETs. (A) The percentage of spontaneously netting neutrophils from healthy controls (HCs) and SVV patients and the percentage of netting neutrophils after irradiation; (B) degree of NET release of neutrophils from HCs and SVV in the presence or absence of aprotinin; (C) degree of NET release of neutrophils after irradiation in the presence or absence of aprotinin. The graph is a minimum of 15 images derived from 3 independent experiments. The error bars of the graph represent SEM. Unpaired Student’s t-test was conducted to analyze the statistical significance of two independent experiments. ***, P<0.001; **, P<0.01; *, P<0.05; NS, not significant.

Article Snippet: A FITC-labeled PNA probe specific for (TTAGGG)n sequences (PANAGENE, Korea) was co-denatured with the slide at 80 °C for 3 min and hybridized at RT for 2 h. Slides were then incubated first with the mouse-anti-human LL37 (HM2070, Hycult biotech, The Netherlands) and rabbit-anti-γH2AX (MABE205, Millipore, USA) antibodies, followed by the secondary Alexa-Fluor-594-labeled goat-anti-mouse antibody and Alexa-Fluor-488-labeled goat-anti-rabbit antibody.

Techniques: Irradiation, Derivative Assay

Journal: Journal of Translational Medicine

Article Title: Conformational changes in myeloperoxidase induced by ubiquitin and NETs containing free ISG15 from systemic lupus erythematosus patients promote a pro-inflammatory cytokine response in CD4 + T cells

doi: 10.1186/s12967-020-02604-5

Figure Lengend Snippet: NETs from SLE patients are enriched in ISG15 and H2B is the main substrate. ISG15 and H2B expression was assessed in spontaneous and LPS induced NETs from SLE patients (n = 6) and healthy controls (n = 3) by indirect immunofluorescence (40X) and confocal microscopy. Spontaneous NETs from a representative SLE patient shows the presence of extracellular ISG15 in the NET ( a ). In contrast to the SLE sample, a representative healthy control image shows the absence of ISG15 in the NET ( b ). Blue (DNA), red (ISG15) and green (H2B). Cumulative data from SLE patients and healthy controls (n = 6 subjects per group) show increased expression of ISG15 in the SLE samples ( c ). In SLE samples, ISG15 and H2B colocalized inside NETs with a high Pearson (R = 0.81) and Costes p value (1.0) in a representative SLE patient ( d ) and boxes represent pooled data (n = 6 subjects per group) that show increased colocalization of SLE samples vs healthy controls ( e ). H2B with ISG15 had the higher colocalization R value compared to other NET proteins such as LL37 or HMGB1 (Additional file : Figures S5 and S6). * p < 0.05

Article Snippet: Additionally, we used mouse anti-human LL37 (LSBio™) or mouse anti-human HMGB1 ( Thermo Fisher™ ). (Additional file : Figures S5 and S6).

Techniques: Expressing, Immunofluorescence, Confocal Microscopy

Journal: Science translational medicine

Article Title: Netting Neutrophils Are Major Inducers of Type I IFN Production in Pediatric Systemic Lupus Erythematosus

doi: 10.1126/scitranslmed.3001201

Figure Lengend Snippet: SLE NETs are loaded with LL37 and HMGB1. (A) Activation with anti-RNP antibodies leads SLE, but not healthy neutrophils, to secrete high levels of LL37 (left) and HMGB1 (right) as quantified by ELISA on culture supernatants (P values were obtained by paired t test). (B) LL37 and HMGB1 colocalize following a globular pattern along SLE NETs. NET release was triggered by treating healthy and SLE neutrophils with PMA (25 nM) for 3 hours. Specimens were then fixed and incubated with antibodies against LL37 and HMGB1 followed by the addition of FITC- or TRITC-conjugated secondary Fab fragments, respectively. DNA was counterstained with Hoechst 33342. Magnification, ×40. This experiment is representative of four independent experiments.

Article Snippet: Specimens were then fixed with 4% paraformaldehyde, and unspecific binding sites were blocked with 5% goat serum in phosphate-buffered saline (PBS) for 30 min. For LL37, HMGB1, and nigrosin-eosin (NE) staining, cover slides were incubated with mouse anti-human LL37 (clone D-5, Santa Cruz Biotechnology), rabbit anti-human HMGB1 (Cell Signaling), or mouse anti-human NE (clone SPM205, Santa Cruz Biotechnology) followed by the appropriate fluorescein isothiocyanate (FITC)– or tetramethyl rhodamine isothiocyanate (TRITC)–conjugated secondary antibody (Fab fragment, all from Santa Cruz Biotechnology).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Science translational medicine

Article Title: Netting Neutrophils Are Major Inducers of Type I IFN Production in Pediatric Systemic Lupus Erythematosus

doi: 10.1126/scitranslmed.3001201

Figure Lengend Snippet: SLE NETs are loaded with LL37 and HMGB1. (A) Activation with anti-RNP antibodies leads SLE, but not healthy neutrophils, to secrete high levels of LL37 (left) and HMGB1 (right) as quantified by ELISA on culture supernatants (P values were obtained by paired t test). (B) LL37 and HMGB1 colocalize following a globular pattern along SLE NETs. NET release was triggered by treating healthy and SLE neutrophils with PMA (25 nM) for 3 hours. Specimens were then fixed and incubated with antibodies against LL37 and HMGB1 followed by the addition of FITC- or TRITC-conjugated secondary Fab fragments, respectively. DNA was counterstained with Hoechst 33342. Magnification, ×40. This experiment is representative of four independent experiments.

Article Snippet: Specimens were then fixed with 4% paraformaldehyde, and unspecific binding sites were blocked with 5% goat serum in phosphate-buffered saline (PBS) for 30 min. For LL37, HMGB1, and nigrosin-eosin (NE) staining, cover slides were incubated with mouse anti-human LL37 (clone D-5, Santa Cruz Biotechnology), rabbit anti-human HMGB1 (Cell Signaling), or mouse anti-human NE (clone SPM205, Santa Cruz Biotechnology) followed by the appropriate fluorescein isothiocyanate (FITC)– or tetramethyl rhodamine isothiocyanate (TRITC)–conjugated secondary antibody (Fab fragment, all from Santa Cruz Biotechnology).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation