y10b antibody  (Santa Cruz Biotechnology)

Journal: EMBO Reports

Article Title: naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation

doi: 10.1038/s44319-024-00150-5

Figure Lengend Snippet: ( A , B ) Confocal microscopy of primary human PMNs stimulated as indicated for 3 h and stained for naRNA (anti-rRNA Y10b, magenta) and DNA (Hoechst 33342, white, n = 3 biological replicates, scale bar: 10 μm, arrowheads point to selected NET strands; representative images in ( A ) were quantified in ( B ) (each dot represents the number of NET-positive tiles in one image quantified from three images/condition). ( C ) Scanning electron microscopy of human primary PMNs treated as indicated and using anti-rRNA primary and immunogold (white arrow)-labeled secondary antibodies and silver enhancement ( n = 3 biological replicates, representative images, scale bars as indicated; the two rightmost images show composite images with signals from secondary electron and backscattered electron detectors for topography and additional material information, respectively). ( D ) RNAseq of PMA NET naRNA ( n = 4 biological replicates) and whole PMN RNA ( n = 1 biological replicate, combined data). ( E ) Quantification of confocal microscopy of primary human PMNs, which were stimulated with NET content (harvested with/without RNase inhibitor and diluted 1:50 or 1:500), and then stained for NETs/DNA using DNA (Hoechst 33342) signal to quantify NET formation ( n = 3 biological replicates, combined data, each dot represents the number of NET-positive tiles in one image quantified from three images/condition). ( F ) as in ( E ) but with/without pre-digestion of NET content with RNase A ( n = 3 biological replicates, representative images, scale bar: 10 μm). ( G ) Quantification of ( F ) ( n = 3 biological replicates, combined data, each dot represents the number of NET-positive tiles in one image quantified from three images/condition). ( H ) As in ( E ) but using purified naRNA ( cf . Fig. ) alone or in complex with exogenously added LL37 ( n = 3 biological replicates, representative images, scale bar: 10 μm). Data information: In ( B ), ( E ), and ( G ), data are presented as mean + SD. * p < 0.05 according to one-way ANOVA. Data shown in ( D ) have been deposited in the NCBI Gene Expression Omnibus under accession number GSE253440. .

Article Snippet: Anti-rRNA (Y10b) , Santa Cruz Biotechnology , sc-33678.

Techniques: Confocal Microscopy, Staining, Electron Microscopy, Labeling, Purification, Gene Expression

Journal: EMBO Reports

Article Title: naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation

doi: 10.1038/s44319-024-00150-5

Figure Lengend Snippet: ( A ) Confocal microscopy of unstimulated or PMA (600 nM) stimulated primary human PMNs after 3 h and stained for naRNA (anti-rRNA Y10b, magenta) and DNA (Hoechst 33342, white). Complete staining and secondary antibody controls only ( n = 3 biological replicates, representative images, scale bar: 10 μm, white arrows indicate NETs). ( B ) Confocal microscopy of unstimulated primary human PMNs (control to Fig. ) after 3 h and stained for naRNA (anti-rRNA Y10b, magenta) and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images, scale bar: 10 μm, white arrows indicate NETs). ( C ) Confocal microscopy of primary murine BM-PMNs of C57BL/6 WT mice stimulated as indicated for 16 h and stained as in ( B ) ( n = 3 biological replicates, representative images, scale bar: 10 μm, white arrows indicate NETs). ( D ) Confocal microscopy of primary human stem cells differentiated in vitro with/without 100 μM 5-ethynyluridine (5-EU), click-labeled with a fluorescent dye (yellow, total RNA), and stained for naRNA (anti-rRNA Y10b, magenta) and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images, scale bar: 10 μm, 2 μm in cropped image, white arrows indicate NETs). ( E ) Brightfield microscopy analysis of control cytospun of primary human stem cell-derived PMNs shown in ( A ) ( n = 3 biological replicates, representative images, scale bar: 10 μm). ( F ) FACS analysis of cells shown in ( D ) and ( E ) ( n = 3 biological replicates, representative data of one biological replicate shown). ( G ) As in ( B ) showing 3D image reconstruction of NETs from z-stacks created with ZenBlue3 ( n = 3 biological replicates, representative images, scale bar as indicated). ( H ) Scanning electron microscopy of PMA-treated human primary PMNs showing only secondary antibody staining (no primary antibody) control of Fig. ( n = 1 biological replicate, representative data; the image on the right is a composite image with signals from secondary electron and backscattered electron detectors for topography and additional material information, respectively). ( I ) Agilent TapeStation quantification of naRNA isolated from mock or PMA NETs (from n = 4–6 biological replicates, combined data, each dot represents one biological replicate). Data information: In ( I ), data are presented as mean + SD. * p < 0.05 according to Mann–Whitney test. Please note that the panel shown in B also appears in Fig. as these two experiments were carried out simultaneously or were part of the same experiment, and hence control conditions (e.g., unstimulated) are identical. .

Article Snippet: Anti-rRNA (Y10b) , Santa Cruz Biotechnology , sc-33678.

Techniques: Confocal Microscopy, Staining, Control, In Vitro, Labeling, Microscopy, Derivative Assay, Electron Microscopy, Isolation, MANN-WHITNEY

Journal: EMBO Reports

Article Title: naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation

doi: 10.1038/s44319-024-00150-5

Figure Lengend Snippet: ( A ) Confocal microscopy of primary human PMNs stimulated as indicated for 3 h and stained for naRNA (anti-rRNA Y10b, magenta), LL37 (anti-hLL37-DyLight550, yellow), and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images, scale bar 2 or 10 μm as indicated). ( B ) Pearson’s correlation coefficient (co-localization) analysis of ( A ) ( n = 3 biological replicates, combined data, each dot represents one image, three images/condition). ( C ) Line plot analysis of LL37, RNA, and DNA staining of primary human PMNs stimulated as indicated in ( A ) was performed using ZenBlue3 software ( n = 3 biological replicates, representative graph, scale bar 2 µm). White arrows indicate co-localization of RNA and LL37. ( D ) As C but staining with SYTO RNAselect instead of anti-rRNA ( n = 3 biological replicates, representative graph, scale bar 10 µm). White arrows indicate co-localization of RNA and LL37. ( E ) As in ( D ) but showing x,z and y,z projections from multiple z-stacks. White arrows indicate co-localization of RNA and LL37. ( F ) As in ( A / C ) but on 50–60 nm ultrathin sections of unstimulated PMNs ( n = 3 biological replicates, representative image, scale bar 2 µm). Line plot analysis was performed using ImageJ-Win64 software ( n = 3 biological replicates, representative graph). White arrows indicate co-localization of RNA and LL37. ( G ) Transmission electron microscopy of unstimulated human primary PMNs using anti-rRNA and anti-hLL-37 primary and immunogold (6 nm (black arrow) and 12 nm (white arrow), respectively)-labeled secondary antibodies ( n = 3 biological replicates, representative images, scale bars as indicated). ( H ) Quantification of confocal microscopy of primary human PMNs stimulated as indicated with fresh or old NETs generated by incubation in PMN culture medium or human serum treatment, respectively, stained for DNA (Hoechst 33342) and quantified as before ( n = 2 biological replicates, each dot represents the number of NET-positive tiles in one image quantified from three images/condition). ( I ) Levels of IL-8, as measured by triplicate ELISA upon release from primary normal human epidermal keratinocytes (NHEK) stimulated for 24 h as indicated in ( F ) ( n = 8 biological replicates, combined data, each dot represents one biological replicate). Data information: In ( B ), ( H ), and ( I ), data are presented as mean + SD. In ( B ) and ( I ), * p < 0.05 according to one-way ANOVA. In ( H ), * p < 0.05 according to Kruskal–Wallis test with Dunn’s correction. Please note that selected panels in ( C ) and ( E ) also appear in Fig. as these two experiments were carried out simultaneously or were part of the same experiment, and hence control conditions (e.g., unstimulated) are identical. .

Article Snippet: Anti-rRNA (Y10b) , Santa Cruz Biotechnology , sc-33678.

Techniques: Confocal Microscopy, Staining, Software, Transmission Assay, Electron Microscopy, Labeling, Generated, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: EMBO Reports

Article Title: naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation

doi: 10.1038/s44319-024-00150-5

Figure Lengend Snippet: ( A ) Confocal microscopy of primary human PMNs left untreated and stained for RNA only (mouse anti-human rRNA Y10b + anti-mouse AF647, magenta), secondary antibody control for RNA and staining for LL37 (anti-mouse AF647 (magenta) + rabbit anti-human LL37-Dylight550 (yellow)) or counterstaining for rRNA Y10b and LL37 (mouse anti-human rRNA Y10b-AF647 (magenta) + rabbit anti-human LL37-Dylight550 (yellow)) and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images, scale bar 10 μm). Controls for Figs. and EV5B. ( B ) Confocal microscopy with line plot analysis of primary human PMNs stimulated as indicated for 3 h and stained for naRNA (anti-rRNA Y10b, magenta), LL37 (anti-hLL37-DyLight550, yellow) and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images). The line plot analysis of LL37, RNA, and DNA staining was performed using ZenBlue3 software. One to two different line plots from the same representative image are shown. Additional examples of images shown in Fig. . ( C ) Confocal microscopy of primary human PMNs left untreated and stained for naRNA (SYTO RNAselect, magenta), LL37 (anti-hLL37-DyLight550, yellow), and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images, scale bar 10 μm). ( D ) Line plot analysis of LL37, RNA, and DNA staining of ( A ). The analysis was performed using ZenBlue3 software. Three different line plots from the same representative image are shown (scale bar 10 μm). Areas of intensity overlap show up as white. ( E ) 3D reconstructions of z-stacks from ( A ). ( F ) Confocal microscopy of PMA-induced NETs from primary human PMNs incubated for 30 min or 4 h with human serum and stained for DNA (Hoechst 33342, white) and naRNA (anti-rRNA Y10b, magenta or red). Lower magnification (left, scale bar = 50 µm) and higher magnification (right, scale bar = 20 µm) for one presentative of n = 2 biological replicates shown. Data information: Please note that selected panels in ( D ) also appear in Fig. , respectively, as these two experiments were carried out simultaneously or were part of the same experiment. .

Article Snippet: Anti-rRNA (Y10b) , Santa Cruz Biotechnology , sc-33678.

Techniques: Confocal Microscopy, Staining, Control, Software, Incubation

Journal: EMBO Reports

Article Title: naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation

doi: 10.1038/s44319-024-00150-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Anti-rRNA (Y10b) , Santa Cruz Biotechnology , sc-33678.

Techniques: Isolation, Derivative Assay, Recombinant, Sequencing, Control, Software, Conjugation Assay, Enzyme-linked Immunosorbent Assay, Microscopy, Imaging, Luciferase, Reporter Assay, Electron Microscopy

Journal: EMBO Reports

Article Title: naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation

doi: 10.1038/s44319-024-00150-5

Figure Lengend Snippet: ( A , B ) Confocal microscopy of primary human PMNs stimulated as indicated for 3 h and stained for naRNA (anti-rRNA Y10b, magenta) and DNA (Hoechst 33342, white, n = 3 biological replicates, scale bar: 10 μm, arrowheads point to selected NET strands; representative images in ( A ) were quantified in ( B ) (each dot represents the number of NET-positive tiles in one image quantified from three images/condition). ( C ) Scanning electron microscopy of human primary PMNs treated as indicated and using anti-rRNA primary and immunogold (white arrow)-labeled secondary antibodies and silver enhancement ( n = 3 biological replicates, representative images, scale bars as indicated; the two rightmost images show composite images with signals from secondary electron and backscattered electron detectors for topography and additional material information, respectively). ( D ) RNAseq of PMA NET naRNA ( n = 4 biological replicates) and whole PMN RNA ( n = 1 biological replicate, combined data). ( E ) Quantification of confocal microscopy of primary human PMNs, which were stimulated with NET content (harvested with/without RNase inhibitor and diluted 1:50 or 1:500), and then stained for NETs/DNA using DNA (Hoechst 33342) signal to quantify NET formation ( n = 3 biological replicates, combined data, each dot represents the number of NET-positive tiles in one image quantified from three images/condition). ( F ) as in ( E ) but with/without pre-digestion of NET content with RNase A ( n = 3 biological replicates, representative images, scale bar: 10 μm). ( G ) Quantification of ( F ) ( n = 3 biological replicates, combined data, each dot represents the number of NET-positive tiles in one image quantified from three images/condition). ( H ) As in ( E ) but using purified naRNA ( cf . Fig. ) alone or in complex with exogenously added LL37 ( n = 3 biological replicates, representative images, scale bar: 10 μm). Data information: In ( B ), ( E ), and ( G ), data are presented as mean + SD. * p < 0.05 according to one-way ANOVA. Data shown in ( D ) have been deposited in the NCBI Gene Expression Omnibus under accession number GSE253440. .

Article Snippet: Anti-rRNA (Y10b) Alexa Fluor® 647 , Santa Cruz Biotechnology , sc-33678 AF647.

Techniques: Confocal Microscopy, Staining, Electron Microscopy, Labeling, Purification, Gene Expression

Journal: EMBO Reports

Article Title: naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation

doi: 10.1038/s44319-024-00150-5

Figure Lengend Snippet: ( A ) Confocal microscopy of unstimulated or PMA (600 nM) stimulated primary human PMNs after 3 h and stained for naRNA (anti-rRNA Y10b, magenta) and DNA (Hoechst 33342, white). Complete staining and secondary antibody controls only ( n = 3 biological replicates, representative images, scale bar: 10 μm, white arrows indicate NETs). ( B ) Confocal microscopy of unstimulated primary human PMNs (control to Fig. ) after 3 h and stained for naRNA (anti-rRNA Y10b, magenta) and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images, scale bar: 10 μm, white arrows indicate NETs). ( C ) Confocal microscopy of primary murine BM-PMNs of C57BL/6 WT mice stimulated as indicated for 16 h and stained as in ( B ) ( n = 3 biological replicates, representative images, scale bar: 10 μm, white arrows indicate NETs). ( D ) Confocal microscopy of primary human stem cells differentiated in vitro with/without 100 μM 5-ethynyluridine (5-EU), click-labeled with a fluorescent dye (yellow, total RNA), and stained for naRNA (anti-rRNA Y10b, magenta) and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images, scale bar: 10 μm, 2 μm in cropped image, white arrows indicate NETs). ( E ) Brightfield microscopy analysis of control cytospun of primary human stem cell-derived PMNs shown in ( A ) ( n = 3 biological replicates, representative images, scale bar: 10 μm). ( F ) FACS analysis of cells shown in ( D ) and ( E ) ( n = 3 biological replicates, representative data of one biological replicate shown). ( G ) As in ( B ) showing 3D image reconstruction of NETs from z-stacks created with ZenBlue3 ( n = 3 biological replicates, representative images, scale bar as indicated). ( H ) Scanning electron microscopy of PMA-treated human primary PMNs showing only secondary antibody staining (no primary antibody) control of Fig. ( n = 1 biological replicate, representative data; the image on the right is a composite image with signals from secondary electron and backscattered electron detectors for topography and additional material information, respectively). ( I ) Agilent TapeStation quantification of naRNA isolated from mock or PMA NETs (from n = 4–6 biological replicates, combined data, each dot represents one biological replicate). Data information: In ( I ), data are presented as mean + SD. * p < 0.05 according to Mann–Whitney test. Please note that the panel shown in B also appears in Fig. as these two experiments were carried out simultaneously or were part of the same experiment, and hence control conditions (e.g., unstimulated) are identical. .

Article Snippet: Anti-rRNA (Y10b) Alexa Fluor® 647 , Santa Cruz Biotechnology , sc-33678 AF647.

Techniques: Confocal Microscopy, Staining, Control, In Vitro, Labeling, Microscopy, Derivative Assay, Electron Microscopy, Isolation, MANN-WHITNEY

Journal: EMBO Reports

Article Title: naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation

doi: 10.1038/s44319-024-00150-5

Figure Lengend Snippet: ( A ) Confocal microscopy of primary human PMNs stimulated as indicated for 3 h and stained for naRNA (anti-rRNA Y10b, magenta), LL37 (anti-hLL37-DyLight550, yellow), and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images, scale bar 2 or 10 μm as indicated). ( B ) Pearson’s correlation coefficient (co-localization) analysis of ( A ) ( n = 3 biological replicates, combined data, each dot represents one image, three images/condition). ( C ) Line plot analysis of LL37, RNA, and DNA staining of primary human PMNs stimulated as indicated in ( A ) was performed using ZenBlue3 software ( n = 3 biological replicates, representative graph, scale bar 2 µm). White arrows indicate co-localization of RNA and LL37. ( D ) As C but staining with SYTO RNAselect instead of anti-rRNA ( n = 3 biological replicates, representative graph, scale bar 10 µm). White arrows indicate co-localization of RNA and LL37. ( E ) As in ( D ) but showing x,z and y,z projections from multiple z-stacks. White arrows indicate co-localization of RNA and LL37. ( F ) As in ( A / C ) but on 50–60 nm ultrathin sections of unstimulated PMNs ( n = 3 biological replicates, representative image, scale bar 2 µm). Line plot analysis was performed using ImageJ-Win64 software ( n = 3 biological replicates, representative graph). White arrows indicate co-localization of RNA and LL37. ( G ) Transmission electron microscopy of unstimulated human primary PMNs using anti-rRNA and anti-hLL-37 primary and immunogold (6 nm (black arrow) and 12 nm (white arrow), respectively)-labeled secondary antibodies ( n = 3 biological replicates, representative images, scale bars as indicated). ( H ) Quantification of confocal microscopy of primary human PMNs stimulated as indicated with fresh or old NETs generated by incubation in PMN culture medium or human serum treatment, respectively, stained for DNA (Hoechst 33342) and quantified as before ( n = 2 biological replicates, each dot represents the number of NET-positive tiles in one image quantified from three images/condition). ( I ) Levels of IL-8, as measured by triplicate ELISA upon release from primary normal human epidermal keratinocytes (NHEK) stimulated for 24 h as indicated in ( F ) ( n = 8 biological replicates, combined data, each dot represents one biological replicate). Data information: In ( B ), ( H ), and ( I ), data are presented as mean + SD. In ( B ) and ( I ), * p < 0.05 according to one-way ANOVA. In ( H ), * p < 0.05 according to Kruskal–Wallis test with Dunn’s correction. Please note that selected panels in ( C ) and ( E ) also appear in Fig. as these two experiments were carried out simultaneously or were part of the same experiment, and hence control conditions (e.g., unstimulated) are identical. .

Article Snippet: Anti-rRNA (Y10b) Alexa Fluor® 647 , Santa Cruz Biotechnology , sc-33678 AF647.

Techniques: Confocal Microscopy, Staining, Software, Transmission Assay, Electron Microscopy, Labeling, Generated, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: EMBO Reports

Article Title: naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation

doi: 10.1038/s44319-024-00150-5

Figure Lengend Snippet: ( A ) Confocal microscopy of primary human PMNs left untreated and stained for RNA only (mouse anti-human rRNA Y10b + anti-mouse AF647, magenta), secondary antibody control for RNA and staining for LL37 (anti-mouse AF647 (magenta) + rabbit anti-human LL37-Dylight550 (yellow)) or counterstaining for rRNA Y10b and LL37 (mouse anti-human rRNA Y10b-AF647 (magenta) + rabbit anti-human LL37-Dylight550 (yellow)) and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images, scale bar 10 μm). Controls for Figs. and EV5B. ( B ) Confocal microscopy with line plot analysis of primary human PMNs stimulated as indicated for 3 h and stained for naRNA (anti-rRNA Y10b, magenta), LL37 (anti-hLL37-DyLight550, yellow) and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images). The line plot analysis of LL37, RNA, and DNA staining was performed using ZenBlue3 software. One to two different line plots from the same representative image are shown. Additional examples of images shown in Fig. . ( C ) Confocal microscopy of primary human PMNs left untreated and stained for naRNA (SYTO RNAselect, magenta), LL37 (anti-hLL37-DyLight550, yellow), and DNA (Hoechst 33342, white, n = 3 biological replicates, representative images, scale bar 10 μm). ( D ) Line plot analysis of LL37, RNA, and DNA staining of ( A ). The analysis was performed using ZenBlue3 software. Three different line plots from the same representative image are shown (scale bar 10 μm). Areas of intensity overlap show up as white. ( E ) 3D reconstructions of z-stacks from ( A ). ( F ) Confocal microscopy of PMA-induced NETs from primary human PMNs incubated for 30 min or 4 h with human serum and stained for DNA (Hoechst 33342, white) and naRNA (anti-rRNA Y10b, magenta or red). Lower magnification (left, scale bar = 50 µm) and higher magnification (right, scale bar = 20 µm) for one presentative of n = 2 biological replicates shown. Data information: Please note that selected panels in ( D ) also appear in Fig. , respectively, as these two experiments were carried out simultaneously or were part of the same experiment. .

Article Snippet: Anti-rRNA (Y10b) Alexa Fluor® 647 , Santa Cruz Biotechnology , sc-33678 AF647.

Techniques: Confocal Microscopy, Staining, Control, Software, Incubation

Journal: EMBO Reports

Article Title: naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation

doi: 10.1038/s44319-024-00150-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Anti-rRNA (Y10b) Alexa Fluor® 647 , Santa Cruz Biotechnology , sc-33678 AF647.

Techniques: Isolation, Derivative Assay, Recombinant, Sequencing, Control, Software, Conjugation Assay, Enzyme-linked Immunosorbent Assay, Microscopy, Imaging, Luciferase, Reporter Assay, Electron Microscopy

Journal: Neuron

Article Title: Customization of the translational complex regulates mRNA-specific translation to control CNS regeneration

doi: 10.1016/j.neuron.2023.06.005

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-rRNA, clone Y10b , Santa Cruz Biotechnology , Cat# sc-33678; RRID: AB_628226.

Techniques: Control, Virus, Recombinant, Protease Inhibitor, Modification, Blocking Assay, Magnetic Beads, Silver Staining, Labeling, Extraction, RNA Extraction, SYBR Green Assay, Bicinchoninic Acid Protein Assay, RNA Sequencing, In Situ Hybridization, shRNA, Luciferase, Plasmid Preparation, Software, Imaging, Microscopy