Structured Review

Carl Roth GmbH hepes
Different buffer tolerances of LILBID-MS and nESI-MS using Avidin and EmrE. Avidin tetramer and EmrE dimer in buffers containing up to 200 mM ammonium acetate were detectable in LILBID-MS ( a , c ) and nESI ( b , d ). <t>TRIS</t> ( a , b ) and <t>HEPES</t> buffer (bottom a – d ) are worse to handle for both instruments, requiring increased laser power and CID voltage with increasing buffer concentrations as indicated. All EmrE buffers contain additionally 5× CMC DDM
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Images

1) Product Images from "LILBID and nESI: Different Native Mass Spectrometry Techniques as Tools in Structural Biology"

Article Title: LILBID and nESI: Different Native Mass Spectrometry Techniques as Tools in Structural Biology

Journal: Journal of the American Society for Mass Spectrometry

doi: 10.1007/s13361-018-2061-4

Different buffer tolerances of LILBID-MS and nESI-MS using Avidin and EmrE. Avidin tetramer and EmrE dimer in buffers containing up to 200 mM ammonium acetate were detectable in LILBID-MS ( a , c ) and nESI ( b , d ). TRIS ( a , b ) and HEPES buffer (bottom a – d ) are worse to handle for both instruments, requiring increased laser power and CID voltage with increasing buffer concentrations as indicated. All EmrE buffers contain additionally 5× CMC DDM
Figure Legend Snippet: Different buffer tolerances of LILBID-MS and nESI-MS using Avidin and EmrE. Avidin tetramer and EmrE dimer in buffers containing up to 200 mM ammonium acetate were detectable in LILBID-MS ( a , c ) and nESI ( b , d ). TRIS ( a , b ) and HEPES buffer (bottom a – d ) are worse to handle for both instruments, requiring increased laser power and CID voltage with increasing buffer concentrations as indicated. All EmrE buffers contain additionally 5× CMC DDM

Techniques Used: Mass Spectrometry, Avidin-Biotin Assay

2) Product Images from "Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)"

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00012

Extracellular NETotic DNA is rich in MPO. Representative fluorescence images of NETs induced by CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) activated in supplement-free RPMI/HEPES. The images show a clear colocalization of MPO (red) with the extracellular DNA-fibers (blue) of released NETs. Scale = 10 μm.
Figure Legend Snippet: Extracellular NETotic DNA is rich in MPO. Representative fluorescence images of NETs induced by CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) activated in supplement-free RPMI/HEPES. The images show a clear colocalization of MPO (red) with the extracellular DNA-fibers (blue) of released NETs. Scale = 10 μm.

Techniques Used: Fluorescence

Influence of serum and serum albumin supplements on NET formation of murine neutrophils. (A) Representative fluorescence images of nuclei of murine neutrophils (Hoechst) after stimulation with CaI (4 μM), PMA (100 nM) or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI/ HEPES without supplements and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS, as indicated. Chromatin decondensation is only inducible in RPMI/ HEPES (white arrow heads). Scale = 50 μm. (B) Percentage of decondensed nuclei/ NETs after stimulation with PMA (100 nM), CaI (4 μM), or LPS (10, 25, or 100 μg/ml) for 180 min. Murine neutrophils were studied in RPMI/ HEPES and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS. Addition of 0.5% BSA or 2% hiFCS to RPMI/ HEPES, inhibits chromatin decondensation induced by PMA, CaI and LPS completely. Error bars = mean ± SEM. ns = not significant. **** p
Figure Legend Snippet: Influence of serum and serum albumin supplements on NET formation of murine neutrophils. (A) Representative fluorescence images of nuclei of murine neutrophils (Hoechst) after stimulation with CaI (4 μM), PMA (100 nM) or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI/ HEPES without supplements and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS, as indicated. Chromatin decondensation is only inducible in RPMI/ HEPES (white arrow heads). Scale = 50 μm. (B) Percentage of decondensed nuclei/ NETs after stimulation with PMA (100 nM), CaI (4 μM), or LPS (10, 25, or 100 μg/ml) for 180 min. Murine neutrophils were studied in RPMI/ HEPES and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS. Addition of 0.5% BSA or 2% hiFCS to RPMI/ HEPES, inhibits chromatin decondensation induced by PMA, CaI and LPS completely. Error bars = mean ± SEM. ns = not significant. **** p

Techniques Used: Fluorescence

Influence of serum and serum albumin supplements on NET formation of human neutrophils. (A) Representative fluorescence images of human neutrophils (chromatin stained by Hoechst) after stimulation with CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI 1,640 with 10 mM HEPES (RPMI/HEPES), RPMI/ HEPES + 0.5% human serum albumin (HSA) or + 1% heat inactivated (56°C) fetal calf serum (hiFCS). Chromatin decondensation induced by PMA is clearly visible with all three culture conditions, while LPS or CaI only cause NET formation in BSA- and HSA-free RPMI/HEPES. Scale = 50 μm. (B) Neutrophils were stimulated to undergo NET formation with CaI (4 μM), PMA (100 nM) or LPS (10, 25, or 100 μg/ml), respectively. Both, HSA and BSA inhibit CaI and LPS-induced formation of NETs (determined as percentage of decondensed nuclei/NETs of total neutrophils). NETosis stimulated by PMA is independent of serum albumin addition. Error bars = mean ± SEM. ns, not significant. * p
Figure Legend Snippet: Influence of serum and serum albumin supplements on NET formation of human neutrophils. (A) Representative fluorescence images of human neutrophils (chromatin stained by Hoechst) after stimulation with CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI 1,640 with 10 mM HEPES (RPMI/HEPES), RPMI/ HEPES + 0.5% human serum albumin (HSA) or + 1% heat inactivated (56°C) fetal calf serum (hiFCS). Chromatin decondensation induced by PMA is clearly visible with all three culture conditions, while LPS or CaI only cause NET formation in BSA- and HSA-free RPMI/HEPES. Scale = 50 μm. (B) Neutrophils were stimulated to undergo NET formation with CaI (4 μM), PMA (100 nM) or LPS (10, 25, or 100 μg/ml), respectively. Both, HSA and BSA inhibit CaI and LPS-induced formation of NETs (determined as percentage of decondensed nuclei/NETs of total neutrophils). NETosis stimulated by PMA is independent of serum albumin addition. Error bars = mean ± SEM. ns, not significant. * p

Techniques Used: Fluorescence, Staining, Albumin Addition

3) Product Images from "LILBID and nESI: Different Native Mass Spectrometry Techniques as Tools in Structural Biology"

Article Title: LILBID and nESI: Different Native Mass Spectrometry Techniques as Tools in Structural Biology

Journal: Journal of the American Society for Mass Spectrometry

doi: 10.1007/s13361-018-2061-4

Different buffer tolerances of LILBID-MS and nESI-MS using Avidin and EmrE. Avidin tetramer and EmrE dimer in buffers containing up to 200 mM ammonium acetate were detectable in LILBID-MS ( a , c ) and nESI ( b , d ). TRIS ( a , b ) and HEPES buffer (bottom a – d ) are worse to handle for both instruments, requiring increased laser power and CID voltage with increasing buffer concentrations as indicated. All EmrE buffers contain additionally 5× CMC DDM
Figure Legend Snippet: Different buffer tolerances of LILBID-MS and nESI-MS using Avidin and EmrE. Avidin tetramer and EmrE dimer in buffers containing up to 200 mM ammonium acetate were detectable in LILBID-MS ( a , c ) and nESI ( b , d ). TRIS ( a , b ) and HEPES buffer (bottom a – d ) are worse to handle for both instruments, requiring increased laser power and CID voltage with increasing buffer concentrations as indicated. All EmrE buffers contain additionally 5× CMC DDM

Techniques Used: Mass Spectrometry, Avidin-Biotin Assay

4) Product Images from "Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue"

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue

Journal: The Journal of Infectious Diseases

doi: 10.1093/infdis/jir434

Effect of calcium on pore conductance in planar lipid bilayers. The panels show histograms (frequency distributions) of the single-channel conductance of PLY pores measured in Ca-free solution or in the presence of 1 or 2 mM CaCl 2 . The membranes were formed from 1% oxidized cholesterol dissolved in n -decane. The aqueous phase contained 100 mM KCl, 10 mM Hepes, and 0.5 μg/mL PLY. The applied voltage was 20 mV; T = 20°C. The histograms suggest virtually unchanged populations of PLY pores with a maximum conductance peak between 20 and 25 nS.
Figure Legend Snippet: Effect of calcium on pore conductance in planar lipid bilayers. The panels show histograms (frequency distributions) of the single-channel conductance of PLY pores measured in Ca-free solution or in the presence of 1 or 2 mM CaCl 2 . The membranes were formed from 1% oxidized cholesterol dissolved in n -decane. The aqueous phase contained 100 mM KCl, 10 mM Hepes, and 0.5 μg/mL PLY. The applied voltage was 20 mV; T = 20°C. The histograms suggest virtually unchanged populations of PLY pores with a maximum conductance peak between 20 and 25 nS.

Techniques Used:

5) Product Images from "Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)"

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00012

Extracellular NETotic DNA is rich in MPO. Representative fluorescence images of NETs induced by CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) activated in supplement-free RPMI/HEPES. The images show a clear colocalization of MPO (red) with the extracellular DNA-fibers (blue) of released NETs. Scale = 10 μm.
Figure Legend Snippet: Extracellular NETotic DNA is rich in MPO. Representative fluorescence images of NETs induced by CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) activated in supplement-free RPMI/HEPES. The images show a clear colocalization of MPO (red) with the extracellular DNA-fibers (blue) of released NETs. Scale = 10 μm.

Techniques Used: Fluorescence

Influence of serum and serum albumin supplements on NET formation of murine neutrophils. (A) Representative fluorescence images of nuclei of murine neutrophils (Hoechst) after stimulation with CaI (4 μM), PMA (100 nM) or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI/ HEPES without supplements and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS, as indicated. Chromatin decondensation is only inducible in RPMI/ HEPES (white arrow heads). Scale = 50 μm. (B) Percentage of decondensed nuclei/ NETs after stimulation with PMA (100 nM), CaI (4 μM), or LPS (10, 25, or 100 μg/ml) for 180 min. Murine neutrophils were studied in RPMI/ HEPES and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS. Addition of 0.5% BSA or 2% hiFCS to RPMI/ HEPES, inhibits chromatin decondensation induced by PMA, CaI and LPS completely. Error bars = mean ± SEM. ns = not significant. **** p
Figure Legend Snippet: Influence of serum and serum albumin supplements on NET formation of murine neutrophils. (A) Representative fluorescence images of nuclei of murine neutrophils (Hoechst) after stimulation with CaI (4 μM), PMA (100 nM) or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI/ HEPES without supplements and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS, as indicated. Chromatin decondensation is only inducible in RPMI/ HEPES (white arrow heads). Scale = 50 μm. (B) Percentage of decondensed nuclei/ NETs after stimulation with PMA (100 nM), CaI (4 μM), or LPS (10, 25, or 100 μg/ml) for 180 min. Murine neutrophils were studied in RPMI/ HEPES and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS. Addition of 0.5% BSA or 2% hiFCS to RPMI/ HEPES, inhibits chromatin decondensation induced by PMA, CaI and LPS completely. Error bars = mean ± SEM. ns = not significant. **** p

Techniques Used: Fluorescence

Influence of serum and serum albumin supplements on NET formation of human neutrophils. (A) Representative fluorescence images of human neutrophils (chromatin stained by Hoechst) after stimulation with CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI 1,640 with 10 mM HEPES (RPMI/HEPES), RPMI/ HEPES + 0.5% human serum albumin (HSA) or + 1% heat inactivated (56°C) fetal calf serum (hiFCS). Chromatin decondensation induced by PMA is clearly visible with all three culture conditions, while LPS or CaI only cause NET formation in BSA- and HSA-free RPMI/HEPES. Scale = 50 μm. (B) Neutrophils were stimulated to undergo NET formation with CaI (4 μM), PMA (100 nM) or LPS (10, 25, or 100 μg/ml), respectively. Both, HSA and BSA inhibit CaI and LPS-induced formation of NETs (determined as percentage of decondensed nuclei/NETs of total neutrophils). NETosis stimulated by PMA is independent of serum albumin addition. Error bars = mean ± SEM. ns, not significant. * p
Figure Legend Snippet: Influence of serum and serum albumin supplements on NET formation of human neutrophils. (A) Representative fluorescence images of human neutrophils (chromatin stained by Hoechst) after stimulation with CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI 1,640 with 10 mM HEPES (RPMI/HEPES), RPMI/ HEPES + 0.5% human serum albumin (HSA) or + 1% heat inactivated (56°C) fetal calf serum (hiFCS). Chromatin decondensation induced by PMA is clearly visible with all three culture conditions, while LPS or CaI only cause NET formation in BSA- and HSA-free RPMI/HEPES. Scale = 50 μm. (B) Neutrophils were stimulated to undergo NET formation with CaI (4 μM), PMA (100 nM) or LPS (10, 25, or 100 μg/ml), respectively. Both, HSA and BSA inhibit CaI and LPS-induced formation of NETs (determined as percentage of decondensed nuclei/NETs of total neutrophils). NETosis stimulated by PMA is independent of serum albumin addition. Error bars = mean ± SEM. ns, not significant. * p

Techniques Used: Fluorescence, Staining, Albumin Addition

6) Product Images from "Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue"

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue

Journal: The Journal of Infectious Diseases

doi: 10.1093/infdis/jir434

Effect of calcium on pore conductance in planar lipid bilayers. The panels show histograms (frequency distributions) of the single-channel conductance of PLY pores measured in Ca-free solution or in the presence of 1 or 2 mM CaCl 2 . The membranes were formed from 1% oxidized cholesterol dissolved in n -decane. The aqueous phase contained 100 mM KCl, 10 mM Hepes, and 0.5 μg/mL PLY. The applied voltage was 20 mV; T = 20°C. The histograms suggest virtually unchanged populations of PLY pores with a maximum conductance peak between 20 and 25 nS.
Figure Legend Snippet: Effect of calcium on pore conductance in planar lipid bilayers. The panels show histograms (frequency distributions) of the single-channel conductance of PLY pores measured in Ca-free solution or in the presence of 1 or 2 mM CaCl 2 . The membranes were formed from 1% oxidized cholesterol dissolved in n -decane. The aqueous phase contained 100 mM KCl, 10 mM Hepes, and 0.5 μg/mL PLY. The applied voltage was 20 mV; T = 20°C. The histograms suggest virtually unchanged populations of PLY pores with a maximum conductance peak between 20 and 25 nS.

Techniques Used:

Related Articles

Centrifugation:

Article Title: DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro
Article Snippet: Briefly, spinal cords of transgenic and non-transgenic embryos were dissected, cut into pieces and pooled in HBSS (Gibco™ /Thermo Fisher Scientific) containing 1% penicillin-streptomycin (Gibco™ /Thermo Fisher Scientific) and 1 M HEPES (Roth, Karlsruhe, BW, Germany), according to the genotype. .. The MNs and glial cells were separated by centrifugation in a 6.2% OptiPrep density gradient solution (Axis-Shield/Alere Technologies AS, Oslo, Norway) diluted in L-15 medium (Sigma-Aldrich).

In Vitro:

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)
Article Snippet: Stimulation Assay Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively. .. 10,000 cells per well were seeded in 96-glassbottom-well-plates (in vitro scientific) for 30 min (37°C, 5% CO2 ) and stimulated to undergo NET formation with either LPS from Pseudomonas aeruginosa (Sigma-Aldrich) at 10, 25, or 100 μg/ml, CaI (Sigma-Aldrich) at 4 μM, or PMA (Sigma-Aldrich) at 100 nM.

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)
Article Snippet: Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively. .. 10,000 cells per well were seeded in 96-glassbottom-well-plates ( in vitro scientific) for 30 min (37°C, 5% CO2 ) and stimulated to undergo NET formation with either LPS from Pseudomonas aeruginosa ) and the percentage of decondensed nuclei/ NETs calculated by Excel (version: 14.3.0; Microsoft corporation).

Modification:

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue
Article Snippet: Cultures, Vital Staining, and Live Imaging Primary astrocytes were prepared from the brains of newborn C57BL/6 mice in a mixed culture as previously reported [ ], grown in Dulbecco’s modified Eagle’s medium (DMEM; GibcoBRL, Invitrogen GmbH, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS; PAN Biotech GmbH, Aidenbach, Germany) and 1% penicillin/streptomycin (GibcoBRL) in 75-cm2 poly-l -ornithine–coated (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) cell culture flasks (Sarstedt AG & Co., Nuembrecht, Germany). .. For live imaging experiments, the cells were incubated in imaging buffer containing 135 mM NaCl, 2 mM CaCl2 , 2.5 mM MgCl2 , 4 mM KCl, and 5 mM Hepes (all from Carl Roth, Karlsruhe, Germany), at a pH of 7.3 at 37°C, with propidium iodide in the medium to stain permeabilized cells and Hoechst 33342 to stain the nuclei of all cells (all stains were diluted 1:1000 from 1 mg/mL stocks, Invitrogen).

Article Title: Speed Switching of Gonococcal Surface Motility Correlates with Proton Motive Force
Article Snippet: .. Before each experiment gonococcal colonies were resuspended in retraction assay medium (RAM) consisting of phenol red-free Dulbecco’s Modified Eagle Medium (GIBCO, Grand Island, NY, USA), 4.5 g/l Glucose (GIBCO), 2 mM L-glutamine (Roth), 8 mM sodium pyruvate (GIBCO), 5 mM ascorbic acid (Roth) and 30 mM HEPES (Roth). ..

Article Title: Motor Properties of PilT-Independent Type 4 Pilus Retraction in Gonococci
Article Snippet: .. In short, all measurements were carried out in retraction assay medium consisting of phenol red-free Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) with 2 mM l -glutamine (Gibco), 8 mM sodium pyruvate (Gibco) and 30 mM HEPES (Roth). .. A suspension of bacteria and carboxylated latex beads with a diameter of 2 μm (Polysciences, Warrington, PA) was applied to a microscope slide and sealed.

Diff-Quik:

Article Title: Chromatin swelling drives neutrophil extracellular trap release
Article Snippet: Cells were counted and suspended at the required concentration for the following procedures with RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) and 0.5% human serum albumin (HSA) (Sigma-Aldrich). .. Cellular identity was confirmed by a cytospin assay (Cytospin 2 Zentrifuge, Shanson) followed by Diff Quick staining (Medion Diagnostics).

Patch Clamp:

Article Title: Mesoscale Architecture Shapes Initiation and Richness of Spontaneous Network Activity
Article Snippet: Paragraph title: Patch-clamp recording and analysis. ... Patch pipettes [6.3 ± 1.4 MΩ (mean and SEM)] were filled with a intracellular solution containing the following: potassium d -gluconate (125 m m ; Sigma-Aldrich), KCl (20 m m ; Sigma-Aldrich), EGTA (5 m m ; Carl Roth), Na2 -ATP (2 m m ; Carl Roth), HEPES (10 m m ; Carl Roth), MgCl2 (2 m m ; Sigma-Aldrich), CaCl2 (0.5 m m ; Sigma-Aldrich), and biocytin (10 mg/ml, Invitrogen), adjusted with KOH to pH 7.4, and with sucrose to 320 mOsm.

Isolation:

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)
Article Snippet: .. Stimulation Assay Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively. .. 10,000 cells per well were seeded in 96-glassbottom-well-plates (in vitro scientific) for 30 min (37°C, 5% CO2 ) and stimulated to undergo NET formation with either LPS from Pseudomonas aeruginosa (Sigma-Aldrich) at 10, 25, or 100 μg/ml, CaI (Sigma-Aldrich) at 4 μM, or PMA (Sigma-Aldrich) at 100 nM.

Article Title: Chromatin swelling drives neutrophil extracellular trap release
Article Snippet: Paragraph title: Isolation of human neutrophils ... Cells were counted and suspended at the required concentration for the following procedures with RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) and 0.5% human serum albumin (HSA) (Sigma-Aldrich).

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)
Article Snippet: .. Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively. .. 10,000 cells per well were seeded in 96-glassbottom-well-plates ( in vitro scientific) for 30 min (37°C, 5% CO2 ) and stimulated to undergo NET formation with either LPS from Pseudomonas aeruginosa ) and the percentage of decondensed nuclei/ NETs calculated by Excel (version: 14.3.0; Microsoft corporation).

Transferring:

Article Title: DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro
Article Snippet: Briefly, spinal cords of transgenic and non-transgenic embryos were dissected, cut into pieces and pooled in HBSS (Gibco™ /Thermo Fisher Scientific) containing 1% penicillin-streptomycin (Gibco™ /Thermo Fisher Scientific) and 1 M HEPES (Roth, Karlsruhe, BW, Germany), according to the genotype. .. The tissue was digested in 0.1% trypsin in HBSS for 15 min at 37°C and triturated with a fire-polished Pasteur pipette subsequent to DNase (AppliChem, Darmstadt, HE, Germany) treatment to obtain a single cell solution.

Microscopy:

Article Title: Motor Properties of PilT-Independent Type 4 Pilus Retraction in Gonococci
Article Snippet: Retraction velocities and stalling forces were measured using an optical tweezers setup assembled on a Zeiss Axiovert 200 microscope ( ). .. In short, all measurements were carried out in retraction assay medium consisting of phenol red-free Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) with 2 mM l -glutamine (Gibco), 8 mM sodium pyruvate (Gibco) and 30 mM HEPES (Roth).

Mouse Assay:

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue
Article Snippet: Acute brain slices were prepared from PD 10–14-day-old C57BL/6 mice by decapitation and vibratome sectioning (Vibroslice NVSL, World Precision Instruments, Berlin, Germany) in artificial cerebrospinal fluid continuously oxygenized with carbogen gas (95% O2 , 5% CO2 ) at 4°C. .. For live imaging experiments, the cells were incubated in imaging buffer containing 135 mM NaCl, 2 mM CaCl2 , 2.5 mM MgCl2 , 4 mM KCl, and 5 mM Hepes (all from Carl Roth, Karlsruhe, Germany), at a pH of 7.3 at 37°C, with propidium iodide in the medium to stain permeabilized cells and Hoechst 33342 to stain the nuclei of all cells (all stains were diluted 1:1000 from 1 mg/mL stocks, Invitrogen).

Article Title: DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro
Article Snippet: Motor neuron-enriched cell culture Pregnant mice were sacrificed by cervical dislocation with subsequent decapitation. .. Briefly, spinal cords of transgenic and non-transgenic embryos were dissected, cut into pieces and pooled in HBSS (Gibco™ /Thermo Fisher Scientific) containing 1% penicillin-streptomycin (Gibco™ /Thermo Fisher Scientific) and 1 M HEPES (Roth, Karlsruhe, BW, Germany), according to the genotype.

Plasmid Preparation:

Article Title: Visualization of Sirtuin 4 Distribution between Mitochondria and the Nucleus, Based on Bimolecular Fluorescence Self-Complementation
Article Snippet: For plasmid expression, chemically competent 10-beta Escherichia coli (E. coli ) cells were obtained from New England Biolabs (Ipswich, MA, USA). .. Agar−Agar Kobe I, CaCl2 , D-Glucose, HEPES, KCl, MgCl2 , NaCl, NaOH, Trypton/Pepton, and yeast extract were purchased from Carl Roth (Graz, Austria).

Transgenic Assay:

Article Title: DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro
Article Snippet: .. Briefly, spinal cords of transgenic and non-transgenic embryos were dissected, cut into pieces and pooled in HBSS (Gibco™ /Thermo Fisher Scientific) containing 1% penicillin-streptomycin (Gibco™ /Thermo Fisher Scientific) and 1 M HEPES (Roth, Karlsruhe, BW, Germany), according to the genotype. .. The tissue was digested in 0.1% trypsin in HBSS for 15 min at 37°C and triturated with a fire-polished Pasteur pipette subsequent to DNase (AppliChem, Darmstadt, HE, Germany) treatment to obtain a single cell solution.

Incubation:

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue
Article Snippet: .. For live imaging experiments, the cells were incubated in imaging buffer containing 135 mM NaCl, 2 mM CaCl2 , 2.5 mM MgCl2 , 4 mM KCl, and 5 mM Hepes (all from Carl Roth, Karlsruhe, Germany), at a pH of 7.3 at 37°C, with propidium iodide in the medium to stain permeabilized cells and Hoechst 33342 to stain the nuclei of all cells (all stains were diluted 1:1000 from 1 mg/mL stocks, Invitrogen). .. Osmolarity was adjusted using NaCl concentration changes.

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)
Article Snippet: Stimulation Assay Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively. .. After an incubation time of 3 h, cells were fixed with 2% PFA (Roth) to end NET formation and stored over night at 4°C.

other:

Article Title: LILBID and nESI: Different Native Mass Spectrometry Techniques as Tools in Structural Biology
Article Snippet: Chemicals TRIS [2-Amino-2-(hydroxymethyl)propan-1,3-diol], HEPES [2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethansulfonsäure], NaCl and KCl was purchased from Carl Roth (Karlsruhe, Germany) in the highest available purity.

Article Title: LILBID and nESI: Different Native Mass Spectrometry Techniques as Tools in Structural Biology
Article Snippet: TRIS [2-Amino-2-(hydroxymethyl)propan-1,3-diol], HEPES [2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethansulfonsäure], NaCl and KCl was purchased from Carl Roth (Karlsruhe, Germany) in the highest available purity.

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue
Article Snippet: The membrane current was measured with a pair of Ag/AgCl electrodes with salt bridges switched in series by a voltage source and a highly sensitive current amplifier (Keithley 427, Keithley Electronics, Garland, TX) in a buffer containing 100 mM KCl, 10 mM Hepes, and various concentrations of CaCl2 (Carl Roth).

Cell Culture:

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue
Article Snippet: Cultures, Vital Staining, and Live Imaging Primary astrocytes were prepared from the brains of newborn C57BL/6 mice in a mixed culture as previously reported [ ], grown in Dulbecco’s modified Eagle’s medium (DMEM; GibcoBRL, Invitrogen GmbH, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS; PAN Biotech GmbH, Aidenbach, Germany) and 1% penicillin/streptomycin (GibcoBRL) in 75-cm2 poly-l -ornithine–coated (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) cell culture flasks (Sarstedt AG & Co., Nuembrecht, Germany). .. For live imaging experiments, the cells were incubated in imaging buffer containing 135 mM NaCl, 2 mM CaCl2 , 2.5 mM MgCl2 , 4 mM KCl, and 5 mM Hepes (all from Carl Roth, Karlsruhe, Germany), at a pH of 7.3 at 37°C, with propidium iodide in the medium to stain permeabilized cells and Hoechst 33342 to stain the nuclei of all cells (all stains were diluted 1:1000 from 1 mg/mL stocks, Invitrogen).

Article Title: DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro
Article Snippet: Paragraph title: Motor neuron-enriched cell culture ... Briefly, spinal cords of transgenic and non-transgenic embryos were dissected, cut into pieces and pooled in HBSS (Gibco™ /Thermo Fisher Scientific) containing 1% penicillin-streptomycin (Gibco™ /Thermo Fisher Scientific) and 1 M HEPES (Roth, Karlsruhe, BW, Germany), according to the genotype.

Expressing:

Article Title: Visualization of Sirtuin 4 Distribution between Mitochondria and the Nucleus, Based on Bimolecular Fluorescence Self-Complementation
Article Snippet: For plasmid expression, chemically competent 10-beta Escherichia coli (E. coli ) cells were obtained from New England Biolabs (Ipswich, MA, USA). .. Agar−Agar Kobe I, CaCl2 , D-Glucose, HEPES, KCl, MgCl2 , NaCl, NaOH, Trypton/Pepton, and yeast extract were purchased from Carl Roth (Graz, Austria).

Staining:

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue
Article Snippet: .. For live imaging experiments, the cells were incubated in imaging buffer containing 135 mM NaCl, 2 mM CaCl2 , 2.5 mM MgCl2 , 4 mM KCl, and 5 mM Hepes (all from Carl Roth, Karlsruhe, Germany), at a pH of 7.3 at 37°C, with propidium iodide in the medium to stain permeabilized cells and Hoechst 33342 to stain the nuclei of all cells (all stains were diluted 1:1000 from 1 mg/mL stocks, Invitrogen). .. Osmolarity was adjusted using NaCl concentration changes.

Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)
Article Snippet: Stimulation Assay Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively. .. The fixed samples were washed with PBS (Sigma-Aldrich) and stained with 1.62 μM Hoechst (Sigma-Aldrich) for 15 min at room temperature.

Article Title: Chromatin swelling drives neutrophil extracellular trap release
Article Snippet: Cells were counted and suspended at the required concentration for the following procedures with RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) and 0.5% human serum albumin (HSA) (Sigma-Aldrich). .. Cellular identity was confirmed by a cytospin assay (Cytospin 2 Zentrifuge, Shanson) followed by Diff Quick staining (Medion Diagnostics).

Concentration Assay:

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue
Article Snippet: For live imaging experiments, the cells were incubated in imaging buffer containing 135 mM NaCl, 2 mM CaCl2 , 2.5 mM MgCl2 , 4 mM KCl, and 5 mM Hepes (all from Carl Roth, Karlsruhe, Germany), at a pH of 7.3 at 37°C, with propidium iodide in the medium to stain permeabilized cells and Hoechst 33342 to stain the nuclei of all cells (all stains were diluted 1:1000 from 1 mg/mL stocks, Invitrogen). .. Osmolarity was adjusted using NaCl concentration changes.

Article Title: Chromatin swelling drives neutrophil extracellular trap release
Article Snippet: .. Cells were counted and suspended at the required concentration for the following procedures with RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) and 0.5% human serum albumin (HSA) (Sigma-Aldrich). .. Experiments with lipopolysaccharides (LPS from Pseudomonas aeruginosa serotype 10.22, strain: ATCC 27316, Sigma-Aldrich) or calcium ionophores (CaI, Sigma-Aldrich) were carried out without addition of 0.5% human serum albumin.

Lysis:

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue
Article Snippet: In these acute slices, cell lysis never exceeded 7% within 12 hours. .. For live imaging experiments, the cells were incubated in imaging buffer containing 135 mM NaCl, 2 mM CaCl2 , 2.5 mM MgCl2 , 4 mM KCl, and 5 mM Hepes (all from Carl Roth, Karlsruhe, Germany), at a pH of 7.3 at 37°C, with propidium iodide in the medium to stain permeabilized cells and Hoechst 33342 to stain the nuclei of all cells (all stains were diluted 1:1000 from 1 mg/mL stocks, Invitrogen).

Software:

Article Title: Mesoscale Architecture Shapes Initiation and Richness of Spontaneous Network Activity
Article Snippet: Patch pipettes [6.3 ± 1.4 MΩ (mean and SEM)] were filled with a intracellular solution containing the following: potassium d -gluconate (125 m m ; Sigma-Aldrich), KCl (20 m m ; Sigma-Aldrich), EGTA (5 m m ; Carl Roth), Na2 -ATP (2 m m ; Carl Roth), HEPES (10 m m ; Carl Roth), MgCl2 (2 m m ; Sigma-Aldrich), CaCl2 (0.5 m m ; Sigma-Aldrich), and biocytin (10 mg/ml, Invitrogen), adjusted with KOH to pH 7.4, and with sucrose to 320 mOsm. .. Data were sampled at 25 kHz (Micro1401 amplifier and Spike2 software; Cambridge Electronics Design).

Derivative Assay:

Article Title: DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro
Article Snippet: MNs derived from spinal cords of E13 mouse embryos were cultured on an astrocytic feeder layer as previously described [ , ]. .. Briefly, spinal cords of transgenic and non-transgenic embryos were dissected, cut into pieces and pooled in HBSS (Gibco™ /Thermo Fisher Scientific) containing 1% penicillin-streptomycin (Gibco™ /Thermo Fisher Scientific) and 1 M HEPES (Roth, Karlsruhe, BW, Germany), according to the genotype.

Imaging:

Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue
Article Snippet: .. For live imaging experiments, the cells were incubated in imaging buffer containing 135 mM NaCl, 2 mM CaCl2 , 2.5 mM MgCl2 , 4 mM KCl, and 5 mM Hepes (all from Carl Roth, Karlsruhe, Germany), at a pH of 7.3 at 37°C, with propidium iodide in the medium to stain permeabilized cells and Hoechst 33342 to stain the nuclei of all cells (all stains were diluted 1:1000 from 1 mg/mL stocks, Invitrogen). .. Osmolarity was adjusted using NaCl concentration changes.

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    Carl Roth GmbH hepes
    Different buffer tolerances of LILBID-MS and nESI-MS using Avidin and EmrE. Avidin tetramer and EmrE dimer in buffers containing up to 200 mM ammonium acetate were detectable in LILBID-MS ( a , c ) and nESI ( b , d ). <t>TRIS</t> ( a , b ) and <t>HEPES</t> buffer (bottom a – d ) are worse to handle for both instruments, requiring increased laser power and CID voltage with increasing buffer concentrations as indicated. All EmrE buffers contain additionally 5× CMC DDM
    Hepes, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Different buffer tolerances of LILBID-MS and nESI-MS using Avidin and EmrE. Avidin tetramer and EmrE dimer in buffers containing up to 200 mM ammonium acetate were detectable in LILBID-MS ( a , c ) and nESI ( b , d ). TRIS ( a , b ) and HEPES buffer (bottom a – d ) are worse to handle for both instruments, requiring increased laser power and CID voltage with increasing buffer concentrations as indicated. All EmrE buffers contain additionally 5× CMC DDM

    Journal: Journal of the American Society for Mass Spectrometry

    Article Title: LILBID and nESI: Different Native Mass Spectrometry Techniques as Tools in Structural Biology

    doi: 10.1007/s13361-018-2061-4

    Figure Lengend Snippet: Different buffer tolerances of LILBID-MS and nESI-MS using Avidin and EmrE. Avidin tetramer and EmrE dimer in buffers containing up to 200 mM ammonium acetate were detectable in LILBID-MS ( a , c ) and nESI ( b , d ). TRIS ( a , b ) and HEPES buffer (bottom a – d ) are worse to handle for both instruments, requiring increased laser power and CID voltage with increasing buffer concentrations as indicated. All EmrE buffers contain additionally 5× CMC DDM

    Article Snippet: Chemicals TRIS [2-Amino-2-(hydroxymethyl)propan-1,3-diol], HEPES [2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethansulfonsäure], NaCl and KCl was purchased from Carl Roth (Karlsruhe, Germany) in the highest available purity.

    Techniques: Mass Spectrometry, Avidin-Biotin Assay

    Extracellular NETotic DNA is rich in MPO. Representative fluorescence images of NETs induced by CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) activated in supplement-free RPMI/HEPES. The images show a clear colocalization of MPO (red) with the extracellular DNA-fibers (blue) of released NETs. Scale = 10 μm.

    Journal: Frontiers in Immunology

    Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

    doi: 10.3389/fimmu.2019.00012

    Figure Lengend Snippet: Extracellular NETotic DNA is rich in MPO. Representative fluorescence images of NETs induced by CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) activated in supplement-free RPMI/HEPES. The images show a clear colocalization of MPO (red) with the extracellular DNA-fibers (blue) of released NETs. Scale = 10 μm.

    Article Snippet: Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively.

    Techniques: Fluorescence

    Influence of serum and serum albumin supplements on NET formation of murine neutrophils. (A) Representative fluorescence images of nuclei of murine neutrophils (Hoechst) after stimulation with CaI (4 μM), PMA (100 nM) or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI/ HEPES without supplements and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS, as indicated. Chromatin decondensation is only inducible in RPMI/ HEPES (white arrow heads). Scale = 50 μm. (B) Percentage of decondensed nuclei/ NETs after stimulation with PMA (100 nM), CaI (4 μM), or LPS (10, 25, or 100 μg/ml) for 180 min. Murine neutrophils were studied in RPMI/ HEPES and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS. Addition of 0.5% BSA or 2% hiFCS to RPMI/ HEPES, inhibits chromatin decondensation induced by PMA, CaI and LPS completely. Error bars = mean ± SEM. ns = not significant. **** p

    Journal: Frontiers in Immunology

    Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

    doi: 10.3389/fimmu.2019.00012

    Figure Lengend Snippet: Influence of serum and serum albumin supplements on NET formation of murine neutrophils. (A) Representative fluorescence images of nuclei of murine neutrophils (Hoechst) after stimulation with CaI (4 μM), PMA (100 nM) or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI/ HEPES without supplements and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS, as indicated. Chromatin decondensation is only inducible in RPMI/ HEPES (white arrow heads). Scale = 50 μm. (B) Percentage of decondensed nuclei/ NETs after stimulation with PMA (100 nM), CaI (4 μM), or LPS (10, 25, or 100 μg/ml) for 180 min. Murine neutrophils were studied in RPMI/ HEPES and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS. Addition of 0.5% BSA or 2% hiFCS to RPMI/ HEPES, inhibits chromatin decondensation induced by PMA, CaI and LPS completely. Error bars = mean ± SEM. ns = not significant. **** p

    Article Snippet: Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively.

    Techniques: Fluorescence

    Influence of serum and serum albumin supplements on NET formation of human neutrophils. (A) Representative fluorescence images of human neutrophils (chromatin stained by Hoechst) after stimulation with CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI 1,640 with 10 mM HEPES (RPMI/HEPES), RPMI/ HEPES + 0.5% human serum albumin (HSA) or + 1% heat inactivated (56°C) fetal calf serum (hiFCS). Chromatin decondensation induced by PMA is clearly visible with all three culture conditions, while LPS or CaI only cause NET formation in BSA- and HSA-free RPMI/HEPES. Scale = 50 μm. (B) Neutrophils were stimulated to undergo NET formation with CaI (4 μM), PMA (100 nM) or LPS (10, 25, or 100 μg/ml), respectively. Both, HSA and BSA inhibit CaI and LPS-induced formation of NETs (determined as percentage of decondensed nuclei/NETs of total neutrophils). NETosis stimulated by PMA is independent of serum albumin addition. Error bars = mean ± SEM. ns, not significant. * p

    Journal: Frontiers in Immunology

    Article Title: Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

    doi: 10.3389/fimmu.2019.00012

    Figure Lengend Snippet: Influence of serum and serum albumin supplements on NET formation of human neutrophils. (A) Representative fluorescence images of human neutrophils (chromatin stained by Hoechst) after stimulation with CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI 1,640 with 10 mM HEPES (RPMI/HEPES), RPMI/ HEPES + 0.5% human serum albumin (HSA) or + 1% heat inactivated (56°C) fetal calf serum (hiFCS). Chromatin decondensation induced by PMA is clearly visible with all three culture conditions, while LPS or CaI only cause NET formation in BSA- and HSA-free RPMI/HEPES. Scale = 50 μm. (B) Neutrophils were stimulated to undergo NET formation with CaI (4 μM), PMA (100 nM) or LPS (10, 25, or 100 μg/ml), respectively. Both, HSA and BSA inhibit CaI and LPS-induced formation of NETs (determined as percentage of decondensed nuclei/NETs of total neutrophils). NETosis stimulated by PMA is independent of serum albumin addition. Error bars = mean ± SEM. ns, not significant. * p

    Article Snippet: Freshly isolated human or murine neutrophils were counted and suspended in RPMI 1640 (Lonza) containing 10 mM HEPES (Roth) (RPMI/HEPES) and 0.5% HSA (Sigma-Aldrich), 0.5%/1%/2% FCS (Biochrom GmbH, Merck Millipore) or 0.5% BSA (Roth), respectively.

    Techniques: Fluorescence, Staining, Albumin Addition

    Different buffer tolerances of LILBID-MS and nESI-MS using Avidin and EmrE. Avidin tetramer and EmrE dimer in buffers containing up to 200 mM ammonium acetate were detectable in LILBID-MS ( a , c ) and nESI ( b , d ). TRIS ( a , b ) and HEPES buffer (bottom a – d ) are worse to handle for both instruments, requiring increased laser power and CID voltage with increasing buffer concentrations as indicated. All EmrE buffers contain additionally 5× CMC DDM

    Journal: Journal of the American Society for Mass Spectrometry

    Article Title: LILBID and nESI: Different Native Mass Spectrometry Techniques as Tools in Structural Biology

    doi: 10.1007/s13361-018-2061-4

    Figure Lengend Snippet: Different buffer tolerances of LILBID-MS and nESI-MS using Avidin and EmrE. Avidin tetramer and EmrE dimer in buffers containing up to 200 mM ammonium acetate were detectable in LILBID-MS ( a , c ) and nESI ( b , d ). TRIS ( a , b ) and HEPES buffer (bottom a – d ) are worse to handle for both instruments, requiring increased laser power and CID voltage with increasing buffer concentrations as indicated. All EmrE buffers contain additionally 5× CMC DDM

    Article Snippet: TRIS [2-Amino-2-(hydroxymethyl)propan-1,3-diol], HEPES [2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethansulfonsäure], NaCl and KCl was purchased from Carl Roth (Karlsruhe, Germany) in the highest available purity.

    Techniques: Mass Spectrometry, Avidin-Biotin Assay

    Effect of calcium on pore conductance in planar lipid bilayers. The panels show histograms (frequency distributions) of the single-channel conductance of PLY pores measured in Ca-free solution or in the presence of 1 or 2 mM CaCl 2 . The membranes were formed from 1% oxidized cholesterol dissolved in n -decane. The aqueous phase contained 100 mM KCl, 10 mM Hepes, and 0.5 μg/mL PLY. The applied voltage was 20 mV; T = 20°C. The histograms suggest virtually unchanged populations of PLY pores with a maximum conductance peak between 20 and 25 nS.

    Journal: The Journal of Infectious Diseases

    Article Title: Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue

    doi: 10.1093/infdis/jir434

    Figure Lengend Snippet: Effect of calcium on pore conductance in planar lipid bilayers. The panels show histograms (frequency distributions) of the single-channel conductance of PLY pores measured in Ca-free solution or in the presence of 1 or 2 mM CaCl 2 . The membranes were formed from 1% oxidized cholesterol dissolved in n -decane. The aqueous phase contained 100 mM KCl, 10 mM Hepes, and 0.5 μg/mL PLY. The applied voltage was 20 mV; T = 20°C. The histograms suggest virtually unchanged populations of PLY pores with a maximum conductance peak between 20 and 25 nS.

    Article Snippet: For live imaging experiments, the cells were incubated in imaging buffer containing 135 mM NaCl, 2 mM CaCl2 , 2.5 mM MgCl2 , 4 mM KCl, and 5 mM Hepes (all from Carl Roth, Karlsruhe, Germany), at a pH of 7.3 at 37°C, with propidium iodide in the medium to stain permeabilized cells and Hoechst 33342 to stain the nuclei of all cells (all stains were diluted 1:1000 from 1 mg/mL stocks, Invitrogen).

    Techniques: