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Biochrom hepes
Hepes, supplied by Biochrom, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 12 article reviews
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hepes - by Bioz Stars, 2020-03
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Related Articles

Clone Assay:

Article Title: Molecular Characterization and Functional Analysis of Murine Interleukin 4 Receptor Allotypes
Article Snippet: Paragraph title: Transfection and Cell Cloning. ... 15 μg of purified plasmid DNA was electroporated into 107 293-EBNA or TF-1 cells in 0.8 ml of medium (Click's RPMI 1640 [Life Technologies, Eggenstein, Germany], 2 mM l -glutamine, 10 mM Hepes, 100 μg/ml penicillin, 60 ng/ml streptomycin, 13 mM NaHCO3 and 5 × 10−5 M 2-ME) supplemented with 10% (vol/vol) FCS (Biochrom, Berlin, Germany) at 900 μF and 260 V in an Easyject electroporation unit (Eurogentec, Seraing, Belgium).

Stable Transfection:

Article Title: Identification of RIP1 as a critical mediator of Smac mimetic-mediated sensitization of glioblastoma cells for Drozitumab-induced apoptosis
Article Snippet: U87MG cells stably expressing cFLIPL or cFLIPS or with stable knockdown of cFLIP have previously been described as well as A172 cells with stable knockdown of TNFR1. .. Briefly, primary glioblastoma cells were isolated by mechanical disaggregation from surgical specimens and cultured in DMEM supplemented with 10% fetal calf serum (Biochrom), 1 mM glutamine (Biochrom), 1% penicillin/streptavidin (Biochrom) and 25 mM HEPES (Biochrom).

Article Title: Inducible shRNA expression for application in a prostate cancer mouse model
Article Snippet: Paragraph title: Cell culture and generation of stable cell pools ... Human prostate carcinoma PC-3 cells were grown in F-12K Kaighn’s modification medium (Gibco-BRL) containing 2 mM glutamine (Gibco-BRL), 20 mM HEPES (Biochrom) and 10% fetal bovine serum (Gibco-BRL).

Article Title: Molecular Characterization and Functional Analysis of Murine Interleukin 4 Receptor Allotypes
Article Snippet: 15 μg of purified plasmid DNA was electroporated into 107 293-EBNA or TF-1 cells in 0.8 ml of medium (Click's RPMI 1640 [Life Technologies, Eggenstein, Germany], 2 mM l -glutamine, 10 mM Hepes, 100 μg/ml penicillin, 60 ng/ml streptomycin, 13 mM NaHCO3 and 5 × 10−5 M 2-ME) supplemented with 10% (vol/vol) FCS (Biochrom, Berlin, Germany) at 900 μF and 260 V in an Easyject electroporation unit (Eurogentec, Seraing, Belgium). .. Stably transfected TF-1 cells were selected by cultivation in the presence of 1 mg/ml G418 (Calbiochem) for 2 wk and subjected to single cell cloning.

Article Title: The Mechanism behind Bacterial Lipoprotein Release: Phenol-Soluble Modulins Mediate Toll-Like Receptor 2 Activation via Extracellular Vesicle Release from Staphylococcus aureus
Article Snippet: HL60 cells stably transfected with human FPR2/ALX have been recently described ( ). .. These cells were grown in RPMI medium (Biochrom) supplemented with 10% FCS (Sigma-Aldrich), 20 mM HEPES (Biochrom), penicillin (100 units/ml), streptomycin (100 µg/ml) (Gibco), 1× GlutaMAX (Gibco), and G418 (Biochrom) at a final concentration of 1 mg/ml.

Suppression Assay:

Article Title: Distinct Kinetics in the Frequency of Peripheral CD4+ T Cells in Patients with Ulcerative Colitis Experiencing a Flare during Treatment with Mesalazine or with a Herbal Preparation of Myrrh, Chamomile, and Coffee Charcoal
Article Snippet: Paragraph title: Suppression assay ... All cells were cultured in Iscove's Modified Dulbecco's Media (IMDM) with GlutaMAX™ (Invitrogen) supplemented with 10% heat-inactivated FCS (PAA Laboratories, Pasching, Austria), 25 mM HEPES (Biochrom, Berlin, Germany), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (both Sigma-Aldrich).

Cytometry:

Article Title: The Mechanism behind Bacterial Lipoprotein Release: Phenol-Soluble Modulins Mediate Toll-Like Receptor 2 Activation via Extracellular Vesicle Release from Staphylococcus aureus
Article Snippet: These cells were grown in RPMI medium (Biochrom) supplemented with 10% FCS (Sigma-Aldrich), 20 mM HEPES (Biochrom), penicillin (100 units/ml), streptomycin (100 µg/ml) (Gibco), 1× GlutaMAX (Gibco), and G418 (Biochrom) at a final concentration of 1 mg/ml. .. Calcium fluxes were analyzed by stimulating cells loaded with Fluo-3-AM (Molecular Probes), and the fluorescence was monitored with a FACSCalibur flow cytometer (Becton, Dickinson), as recently described ( ).

Construct:

Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids
Article Snippet: Paragraph title: Cells, reagents and plasmid constructs ... PHA-stimulated PBMCs (PHA lymphoblasts) were prepared by cultivating human PBMCs for 7 days in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml, 10 mM HEPES and 0.5 μ g/ml PHA (Biochrom, Berlin, Germany).

Incubation:

Article Title: Rad51 Accumulation at Sites of DNA Damage and in Postreplicative Chromatin
Article Snippet: Microirradiation Using a UVA Pulse Laser For microirradiation, cells were seeded on round coverslips (Schubert & Weiss) and incubated with medium containing BrdU for 20 h. Before microirradiation the coverslips were mounted in a living cell chamber model FCS2 (Bioptechs). .. During microirradiation, the cells were kept in RPMI medium containing 25 mM Hepes and 10% FCS (Biochrom).

Expressing:

Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids
Article Snippet: PHA-stimulated PBMCs (PHA lymphoblasts) were prepared by cultivating human PBMCs for 7 days in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml, 10 mM HEPES and 0.5 μ g/ml PHA (Biochrom, Berlin, Germany). .. The same medium was used for murine NIH3T3 fibroblasts expressing α ν β 3 integrin, which were described previously.

Article Title: Identification of RIP1 as a critical mediator of Smac mimetic-mediated sensitization of glioblastoma cells for Drozitumab-induced apoptosis
Article Snippet: U87MG cells stably expressing cFLIPL or cFLIPS or with stable knockdown of cFLIP have previously been described as well as A172 cells with stable knockdown of TNFR1. .. Briefly, primary glioblastoma cells were isolated by mechanical disaggregation from surgical specimens and cultured in DMEM supplemented with 10% fetal calf serum (Biochrom), 1 mM glutamine (Biochrom), 1% penicillin/streptavidin (Biochrom) and 25 mM HEPES (Biochrom).

Article Title: Inducible shRNA expression for application in a prostate cancer mouse model
Article Snippet: Human prostate carcinoma PC-3 cells were grown in F-12K Kaighn’s modification medium (Gibco-BRL) containing 2 mM glutamine (Gibco-BRL), 20 mM HEPES (Biochrom) and 10% fetal bovine serum (Gibco-BRL). .. The shRNA expression plasmids and the TetR expression plasmid (pcDNA™6/TR) were transfected with Effectene transfection reagent (Qiagen).

Modification:

Article Title: Inducible shRNA expression for application in a prostate cancer mouse model
Article Snippet: .. Human prostate carcinoma PC-3 cells were grown in F-12K Kaighn’s modification medium (Gibco-BRL) containing 2 mM glutamine (Gibco-BRL), 20 mM HEPES (Biochrom) and 10% fetal bovine serum (Gibco-BRL). .. The shRNA expression plasmids and the TetR expression plasmid (pcDNA™6/TR) were transfected with Effectene transfection reagent (Qiagen).

Article Title: Distinct Kinetics in the Frequency of Peripheral CD4+ T Cells in Patients with Ulcerative Colitis Experiencing a Flare during Treatment with Mesalazine or with a Herbal Preparation of Myrrh, Chamomile, and Coffee Charcoal
Article Snippet: .. All cells were cultured in Iscove's Modified Dulbecco's Media (IMDM) with GlutaMAX™ (Invitrogen) supplemented with 10% heat-inactivated FCS (PAA Laboratories, Pasching, Austria), 25 mM HEPES (Biochrom, Berlin, Germany), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (both Sigma-Aldrich). ..

Article Title: Distinct Shift in Beta-Cell Glutaredoxin 5 Expression Is Mediated by Hypoxia and Lipotoxicity Both In Vivo and In Vitro
Article Snippet: .. Mouse insulinoma cells 6th subclone (MIN6 cells) cell line was obtained from Dr. Sigurd Lenzen (Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany) ( ) (originally from Dr. Miyazaki, Institute for Medical Genetics, Kumamoto University Medical School, Japan ( )) and cells were routinely maintained in Dulbecco’s modified Eagle medium (DMEM, Life Technologies, Darmstadt, Germany) containing 25 mM glucose, supplemented with 10% fetal calf serum (biowest, Nuaillé, France), 2 mM l -glutamine, 25 mM Hepes (Biochrom, Berlin, Germany), 285 µM 2-mercaptoethanol (Life Technologies, Darmstadt, Germany), and 1% penicillin/streptomycin (Life Technologies, Darmstadt, Germany). ..

Electroporation:

Article Title: Molecular Characterization and Functional Analysis of Murine Interleukin 4 Receptor Allotypes
Article Snippet: .. 15 μg of purified plasmid DNA was electroporated into 107 293-EBNA or TF-1 cells in 0.8 ml of medium (Click's RPMI 1640 [Life Technologies, Eggenstein, Germany], 2 mM l -glutamine, 10 mM Hepes, 100 μg/ml penicillin, 60 ng/ml streptomycin, 13 mM NaHCO3 and 5 × 10−5 M 2-ME) supplemented with 10% (vol/vol) FCS (Biochrom, Berlin, Germany) at 900 μF and 260 V in an Easyject electroporation unit (Eurogentec, Seraing, Belgium). .. The selection of sIL-4R–transfected 293-EBNA cells was started after 36 h by addition of hygromycin (Calbiochem, Bad Soden, Germany) to a final concentration of 0.3 mg/ml.

Transfection:

Article Title: Inducible shRNA expression for application in a prostate cancer mouse model
Article Snippet: Human prostate carcinoma PC-3 cells were grown in F-12K Kaighn’s modification medium (Gibco-BRL) containing 2 mM glutamine (Gibco-BRL), 20 mM HEPES (Biochrom) and 10% fetal bovine serum (Gibco-BRL). .. The shRNA expression plasmids and the TetR expression plasmid (pcDNA™6/TR) were transfected with Effectene transfection reagent (Qiagen).

Article Title: Molecular Characterization and Functional Analysis of Murine Interleukin 4 Receptor Allotypes
Article Snippet: Paragraph title: Transfection and Cell Cloning. ... 15 μg of purified plasmid DNA was electroporated into 107 293-EBNA or TF-1 cells in 0.8 ml of medium (Click's RPMI 1640 [Life Technologies, Eggenstein, Germany], 2 mM l -glutamine, 10 mM Hepes, 100 μg/ml penicillin, 60 ng/ml streptomycin, 13 mM NaHCO3 and 5 × 10−5 M 2-ME) supplemented with 10% (vol/vol) FCS (Biochrom, Berlin, Germany) at 900 μF and 260 V in an Easyject electroporation unit (Eurogentec, Seraing, Belgium).

Article Title: The Mechanism behind Bacterial Lipoprotein Release: Phenol-Soluble Modulins Mediate Toll-Like Receptor 2 Activation via Extracellular Vesicle Release from Staphylococcus aureus
Article Snippet: HL60 cells stably transfected with human FPR2/ALX have been recently described ( ). .. These cells were grown in RPMI medium (Biochrom) supplemented with 10% FCS (Sigma-Aldrich), 20 mM HEPES (Biochrom), penicillin (100 units/ml), streptomycin (100 µg/ml) (Gibco), 1× GlutaMAX (Gibco), and G418 (Biochrom) at a final concentration of 1 mg/ml.

Cell Culture:

Article Title: Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function
Article Snippet: Paragraph title: Cells and Cell Culture ... Muscle specimens were placed in 30.2 mM HEPES containing 130 mM NaCl, 3 mM KCl, 10 mM D-Glucose and subsequently digested in 254 U/ml Collagenase CLS II (Biochrom AG, Berlin, Germany), 100 U/ml Dispase II (Roche, Grenzach-W´yhlen, Germany) and trypsin/EDTA at 37°C for 45 minutes.

Article Title: Ultrafast in cellulo photoinduced dynamics processes of the paradigm molecular light switch [Ru(bpy)2dppz]2+
Article Snippet: The cells were cultivated in RPMI 1640 liquid medium with 20 mM HEPES, stable glutamine (FG 1235, Biochrom AG, Germany), 10% fetal bovine serum (10099-133, Life Technologies, Germany) together with 100 units/mL of penicillin and 100 μ g/mL of streptomycin (15140, Gibco® , Life Technologies GmbH, Germany). .. The 75 cm2 cell culture flasks (658170; Greiner Bio-One GmbH, Germany) were used for cultivation.

Article Title: Overexpression of Inosine 5?-Monophosphate Dehydrogenase Type II Mediates Chemoresistance to Human Osteosarcoma Cells
Article Snippet: Paragraph title: Cell culture and drug treatment ... Cells were maintained in RPMI 1640 (Lonza GmbH, Wuppertal, Germany) containing 2 mM L-glutamine and 25 mM HEPES and supplemented with fetal calf serum (Biochrom, Berlin, Germany), and 100 U/ml penicillin/streptomycin (Lonza GmbH) at 37°C in a humidified 5% CO2 atmosphere.

Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids
Article Snippet: THP-1 and U937 cells obtained from ATCC were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml and 10 mM HEPES (all from Invitrogen Life Technologies, Karlsruhe, Germany). .. PHA-stimulated PBMCs (PHA lymphoblasts) were prepared by cultivating human PBMCs for 7 days in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml, 10 mM HEPES and 0.5 μ g/ml PHA (Biochrom, Berlin, Germany).

Article Title: Identification of RIP1 as a critical mediator of Smac mimetic-mediated sensitization of glioblastoma cells for Drozitumab-induced apoptosis
Article Snippet: .. Briefly, primary glioblastoma cells were isolated by mechanical disaggregation from surgical specimens and cultured in DMEM supplemented with 10% fetal calf serum (Biochrom), 1 mM glutamine (Biochrom), 1% penicillin/streptavidin (Biochrom) and 25 mM HEPES (Biochrom). ..

Article Title: Inducible shRNA expression for application in a prostate cancer mouse model
Article Snippet: Paragraph title: Cell culture and generation of stable cell pools ... Human prostate carcinoma PC-3 cells were grown in F-12K Kaighn’s modification medium (Gibco-BRL) containing 2 mM glutamine (Gibco-BRL), 20 mM HEPES (Biochrom) and 10% fetal bovine serum (Gibco-BRL).

Article Title: Distinct Kinetics in the Frequency of Peripheral CD4+ T Cells in Patients with Ulcerative Colitis Experiencing a Flare during Treatment with Mesalazine or with a Herbal Preparation of Myrrh, Chamomile, and Coffee Charcoal
Article Snippet: .. All cells were cultured in Iscove's Modified Dulbecco's Media (IMDM) with GlutaMAX™ (Invitrogen) supplemented with 10% heat-inactivated FCS (PAA Laboratories, Pasching, Austria), 25 mM HEPES (Biochrom, Berlin, Germany), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (both Sigma-Aldrich). ..

Article Title: Distinct Shift in Beta-Cell Glutaredoxin 5 Expression Is Mediated by Hypoxia and Lipotoxicity Both In Vivo and In Vitro
Article Snippet: Paragraph title: 2.5. Cell Culture and Protein Analysis ... Mouse insulinoma cells 6th subclone (MIN6 cells) cell line was obtained from Dr. Sigurd Lenzen (Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany) ( ) (originally from Dr. Miyazaki, Institute for Medical Genetics, Kumamoto University Medical School, Japan ( )) and cells were routinely maintained in Dulbecco’s modified Eagle medium (DMEM, Life Technologies, Darmstadt, Germany) containing 25 mM glucose, supplemented with 10% fetal calf serum (biowest, Nuaillé, France), 2 mM l -glutamine, 25 mM Hepes (Biochrom, Berlin, Germany), 285 µM 2-mercaptoethanol (Life Technologies, Darmstadt, Germany), and 1% penicillin/streptomycin (Life Technologies, Darmstadt, Germany).

Article Title: Conjugation of Hydroxyethyl Starch to Desferrioxamine (DFO) Modulates the Dual Role of DFO in Yersinia enterocolitica Infection
Article Snippet: .. Cells were cultured in Click/RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 2 mM l -glutamine (Life Technologies GIBCO BRL, Berlin, Germany), 10 mM HEPES (Biochrom), 5 × 10−5 M 2-mercaptoethanol, 100 μg of streptomycin per ml, 100 U of penicillin (Biochrom) per ml, and 10% heat-inactivated fetal calf serum (Biochrom). .. Spleens from mice were removed aseptically, and single-cell suspensions were prepared; 2 × 105 splenic mononuclear cells (SMNC) were cultivated in round-bottom microtiter plates (Nunc, Wiesbaden, Germany) and incubated with serial dilutions of either DFO, HES-DFO, FO, or HES-FO.

Article Title: Herpes simplex virus thymidine kinase/ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases
Article Snippet: .. The human neuroblastoma cell line SH-EP was cultured in RPMI 1640 medium (Life Technologies, Eggenstein, Germany) supplemented with 10% heat-inactivated FCS (Biochrom, Berlin), 10 mM of Hepes, pH 7.3 (Biochrom), 100 units/ml of penicillin, 100 μg/ml of streptomycin (both from Life Technologies), and 2 mM of l -glutamine (Biochrom). .. The retroviral vector tgLs(+)HyTK, which contains a hygromycin phosphotransferase–TK fusion gene driven by the long terminal repeat promoter , was employed.

Generated:

Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids
Article Snippet: Monocyte-derived macrophages were generated by differentiation of peripheral monocytes with 20 ng/ml M-CSF or GM-CSF (R & D Systems, Heidelberg, Germany) in macrophage medium (Invitrogen Life Technologies) supplemented with 5% autologous serum for 7 days. .. PHA-stimulated PBMCs (PHA lymphoblasts) were prepared by cultivating human PBMCs for 7 days in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml, 10 mM HEPES and 0.5 μ g/ml PHA (Biochrom, Berlin, Germany).

Injection:

Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids
Article Snippet: PHA-stimulated PBMCs (PHA lymphoblasts) were prepared by cultivating human PBMCs for 7 days in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml, 10 mM HEPES and 0.5 μ g/ml PHA (Biochrom, Berlin, Germany). .. Thioglycollate-elicited macrophages were isolated from C57BL/6 and MFG-E8−/− mice by peritoneal lavage of mice that had been injected with 1 ml of 2% thioglycollate in PBS 3 days before.

Article Title: Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4+ Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression
Article Snippet: Preparation and Culture of Cells and Lavage VLE RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (FCS, Biochrom), 10 mM HEPES (Biochrom), 2 mM glutamine, 0.06 mg/ml penicillin, 0.02 mg/ml gentamicin, and 0.05 mM 2-Mercapthoethanol (all from Sigma-Aldrich, Taufkirchen, Germany) was used as culture medium (CM). .. To collect the peritoneal lavage and cells, 3 ml PBS were injected into the peritoneal cavity, aspirated, and centrifuged.

Fluorescence:

Article Title: The Mechanism behind Bacterial Lipoprotein Release: Phenol-Soluble Modulins Mediate Toll-Like Receptor 2 Activation via Extracellular Vesicle Release from Staphylococcus aureus
Article Snippet: These cells were grown in RPMI medium (Biochrom) supplemented with 10% FCS (Sigma-Aldrich), 20 mM HEPES (Biochrom), penicillin (100 units/ml), streptomycin (100 µg/ml) (Gibco), 1× GlutaMAX (Gibco), and G418 (Biochrom) at a final concentration of 1 mg/ml. .. Calcium fluxes were analyzed by stimulating cells loaded with Fluo-3-AM (Molecular Probes), and the fluorescence was monitored with a FACSCalibur flow cytometer (Becton, Dickinson), as recently described ( ).

Magnetic Beads:

Article Title: Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function
Article Snippet: Muscle specimens were placed in 30.2 mM HEPES containing 130 mM NaCl, 3 mM KCl, 10 mM D-Glucose and subsequently digested in 254 U/ml Collagenase CLS II (Biochrom AG, Berlin, Germany), 100 U/ml Dispase II (Roche, Grenzach-W´yhlen, Germany) and trypsin/EDTA at 37°C for 45 minutes. .. Myoblasts were purified using anti-CD56 ab-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany).

Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids
Article Snippet: Primary human monocytes were positively selected from PBMCs with anti-CD14 magnetic beads (Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer's recommendation. .. PHA-stimulated PBMCs (PHA lymphoblasts) were prepared by cultivating human PBMCs for 7 days in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml, 10 mM HEPES and 0.5 μ g/ml PHA (Biochrom, Berlin, Germany).

Isolation:

Article Title: Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function
Article Snippet: Most experiments on human myotubes were performed with primary human muscle cells isolated from healthy probands and from patients with LGMD2B: DYSF c.855+1delG and c.895G > A; p.G299R; homozygosity for DYSF c.4022T > C; p.L1341P. .. Muscle specimens were placed in 30.2 mM HEPES containing 130 mM NaCl, 3 mM KCl, 10 mM D-Glucose and subsequently digested in 254 U/ml Collagenase CLS II (Biochrom AG, Berlin, Germany), 100 U/ml Dispase II (Roche, Grenzach-W´yhlen, Germany) and trypsin/EDTA at 37°C for 45 minutes.

Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids
Article Snippet: PHA-stimulated PBMCs (PHA lymphoblasts) were prepared by cultivating human PBMCs for 7 days in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml, 10 mM HEPES and 0.5 μ g/ml PHA (Biochrom, Berlin, Germany). .. Thioglycollate-elicited macrophages were isolated from C57BL/6 and MFG-E8−/− mice by peritoneal lavage of mice that had been injected with 1 ml of 2% thioglycollate in PBS 3 days before.

Article Title: Identification of RIP1 as a critical mediator of Smac mimetic-mediated sensitization of glioblastoma cells for Drozitumab-induced apoptosis
Article Snippet: .. Briefly, primary glioblastoma cells were isolated by mechanical disaggregation from surgical specimens and cultured in DMEM supplemented with 10% fetal calf serum (Biochrom), 1 mM glutamine (Biochrom), 1% penicillin/streptavidin (Biochrom) and 25 mM HEPES (Biochrom). ..

Article Title: Early Production of the Neutrophil-Derived Lipid Mediators LTB4 and LXA4 Is Modulated by Intracellular Infection with Leishmania major
Article Snippet: .. After isolation neutrophils were resuspended in complete medium (RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum) and 50 μ M β -mercaptoethanol (all from Sigma-Aldrich, Steinheim, Germany), 4 mM L-glutamine, and 10 mM HEPES (both Biochrom, Berlin, Germany). .. The obtained cell preparations contained > 99% granulocytes according to morphological examination of cytocentrifuge slides stained with Diff Quik (Medion Diagnostics, Düdingen, Switzerland).

Flow Cytometry:

Article Title: The Mechanism behind Bacterial Lipoprotein Release: Phenol-Soluble Modulins Mediate Toll-Like Receptor 2 Activation via Extracellular Vesicle Release from Staphylococcus aureus
Article Snippet: These cells were grown in RPMI medium (Biochrom) supplemented with 10% FCS (Sigma-Aldrich), 20 mM HEPES (Biochrom), penicillin (100 units/ml), streptomycin (100 µg/ml) (Gibco), 1× GlutaMAX (Gibco), and G418 (Biochrom) at a final concentration of 1 mg/ml. .. Calcium fluxes were analyzed by stimulating cells loaded with Fluo-3-AM (Molecular Probes), and the fluorescence was monitored with a FACSCalibur flow cytometer (Becton, Dickinson), as recently described ( ).

Labeling:

Article Title: Distinct Kinetics in the Frequency of Peripheral CD4+ T Cells in Patients with Ulcerative Colitis Experiencing a Flare during Treatment with Mesalazine or with a Herbal Preparation of Myrrh, Chamomile, and Coffee Charcoal
Article Snippet: CD4+ CD25− responder T cells (3–4×104 , depending on the blood donor) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) and either cultured alone or co-cultured with CD4+ CD25high regulatory T cells (1.5–2×104 ) from the same donor at a ratio of 2 to 1 in the presence of Treg Suppression Inspector (Miltenyi Biotec) for 3 days. .. All cells were cultured in Iscove's Modified Dulbecco's Media (IMDM) with GlutaMAX™ (Invitrogen) supplemented with 10% heat-inactivated FCS (PAA Laboratories, Pasching, Austria), 25 mM HEPES (Biochrom, Berlin, Germany), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (both Sigma-Aldrich).

Mouse Assay:

Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids
Article Snippet: PHA-stimulated PBMCs (PHA lymphoblasts) were prepared by cultivating human PBMCs for 7 days in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml, 10 mM HEPES and 0.5 μ g/ml PHA (Biochrom, Berlin, Germany). .. Thioglycollate-elicited macrophages were isolated from C57BL/6 and MFG-E8−/− mice by peritoneal lavage of mice that had been injected with 1 ml of 2% thioglycollate in PBS 3 days before.

Diff-Quik:

Article Title: Early Production of the Neutrophil-Derived Lipid Mediators LTB4 and LXA4 Is Modulated by Intracellular Infection with Leishmania major
Article Snippet: After isolation neutrophils were resuspended in complete medium (RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum) and 50 μ M β -mercaptoethanol (all from Sigma-Aldrich, Steinheim, Germany), 4 mM L-glutamine, and 10 mM HEPES (both Biochrom, Berlin, Germany). .. The obtained cell preparations contained > 99% granulocytes according to morphological examination of cytocentrifuge slides stained with Diff Quik (Medion Diagnostics, Düdingen, Switzerland).

shRNA:

Article Title: Inducible shRNA expression for application in a prostate cancer mouse model
Article Snippet: Human prostate carcinoma PC-3 cells were grown in F-12K Kaighn’s modification medium (Gibco-BRL) containing 2 mM glutamine (Gibco-BRL), 20 mM HEPES (Biochrom) and 10% fetal bovine serum (Gibco-BRL). .. The shRNA expression plasmids and the TetR expression plasmid (pcDNA™6/TR) were transfected with Effectene transfection reagent (Qiagen).

Purification:

Article Title: Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function
Article Snippet: Muscle specimens were placed in 30.2 mM HEPES containing 130 mM NaCl, 3 mM KCl, 10 mM D-Glucose and subsequently digested in 254 U/ml Collagenase CLS II (Biochrom AG, Berlin, Germany), 100 U/ml Dispase II (Roche, Grenzach-W´yhlen, Germany) and trypsin/EDTA at 37°C for 45 minutes. .. Myoblasts were purified using anti-CD56 ab-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany).

Article Title: Molecular Characterization and Functional Analysis of Murine Interleukin 4 Receptor Allotypes
Article Snippet: .. 15 μg of purified plasmid DNA was electroporated into 107 293-EBNA or TF-1 cells in 0.8 ml of medium (Click's RPMI 1640 [Life Technologies, Eggenstein, Germany], 2 mM l -glutamine, 10 mM Hepes, 100 μg/ml penicillin, 60 ng/ml streptomycin, 13 mM NaHCO3 and 5 × 10−5 M 2-ME) supplemented with 10% (vol/vol) FCS (Biochrom, Berlin, Germany) at 900 μF and 260 V in an Easyject electroporation unit (Eurogentec, Seraing, Belgium). .. The selection of sIL-4R–transfected 293-EBNA cells was started after 36 h by addition of hygromycin (Calbiochem, Bad Soden, Germany) to a final concentration of 0.3 mg/ml.

Article Title: Early Production of the Neutrophil-Derived Lipid Mediators LTB4 and LXA4 Is Modulated by Intracellular Infection with Leishmania major
Article Snippet: Using layered lymphocyte separation medium 1077 (PAA, Pasching, Austria) and Histopaque 1119 (Sigma-Aldrich, Deisenhofen, Germany) first, the separated granulocytes were purified in a discontinuous Percoll (Amersham Biosciences, Uppsala, Sweden) gradient centrifugation. .. After isolation neutrophils were resuspended in complete medium (RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum) and 50 μ M β -mercaptoethanol (all from Sigma-Aldrich, Steinheim, Germany), 4 mM L-glutamine, and 10 mM HEPES (both Biochrom, Berlin, Germany).

Plasmid Preparation:

Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids
Article Snippet: Paragraph title: Cells, reagents and plasmid constructs ... PHA-stimulated PBMCs (PHA lymphoblasts) were prepared by cultivating human PBMCs for 7 days in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml, 10 mM HEPES and 0.5 μ g/ml PHA (Biochrom, Berlin, Germany).

Article Title: Inducible shRNA expression for application in a prostate cancer mouse model
Article Snippet: Human prostate carcinoma PC-3 cells were grown in F-12K Kaighn’s modification medium (Gibco-BRL) containing 2 mM glutamine (Gibco-BRL), 20 mM HEPES (Biochrom) and 10% fetal bovine serum (Gibco-BRL). .. The shRNA expression plasmids and the TetR expression plasmid (pcDNA™6/TR) were transfected with Effectene transfection reagent (Qiagen).

Article Title: Molecular Characterization and Functional Analysis of Murine Interleukin 4 Receptor Allotypes
Article Snippet: .. 15 μg of purified plasmid DNA was electroporated into 107 293-EBNA or TF-1 cells in 0.8 ml of medium (Click's RPMI 1640 [Life Technologies, Eggenstein, Germany], 2 mM l -glutamine, 10 mM Hepes, 100 μg/ml penicillin, 60 ng/ml streptomycin, 13 mM NaHCO3 and 5 × 10−5 M 2-ME) supplemented with 10% (vol/vol) FCS (Biochrom, Berlin, Germany) at 900 μF and 260 V in an Easyject electroporation unit (Eurogentec, Seraing, Belgium). .. The selection of sIL-4R–transfected 293-EBNA cells was started after 36 h by addition of hygromycin (Calbiochem, Bad Soden, Germany) to a final concentration of 0.3 mg/ml.

Selection:

Article Title: Inducible shRNA expression for application in a prostate cancer mouse model
Article Snippet: Human prostate carcinoma PC-3 cells were grown in F-12K Kaighn’s modification medium (Gibco-BRL) containing 2 mM glutamine (Gibco-BRL), 20 mM HEPES (Biochrom) and 10% fetal bovine serum (Gibco-BRL). .. PC-3 cells stably expressing siRNA were established by selection with medium containing geneticin (600 µg/ml; Invitrogen) alone or in the case of double transfection experiments with pcDNA™6/TR in combination with blasticidin (10 µg/ml).

Article Title: Molecular Characterization and Functional Analysis of Murine Interleukin 4 Receptor Allotypes
Article Snippet: 15 μg of purified plasmid DNA was electroporated into 107 293-EBNA or TF-1 cells in 0.8 ml of medium (Click's RPMI 1640 [Life Technologies, Eggenstein, Germany], 2 mM l -glutamine, 10 mM Hepes, 100 μg/ml penicillin, 60 ng/ml streptomycin, 13 mM NaHCO3 and 5 × 10−5 M 2-ME) supplemented with 10% (vol/vol) FCS (Biochrom, Berlin, Germany) at 900 μF and 260 V in an Easyject electroporation unit (Eurogentec, Seraing, Belgium). .. The selection of sIL-4R–transfected 293-EBNA cells was started after 36 h by addition of hygromycin (Calbiochem, Bad Soden, Germany) to a final concentration of 0.3 mg/ml.

Sample Prep:

Article Title: Ultrafast in cellulo photoinduced dynamics processes of the paradigm molecular light switch [Ru(bpy)2dppz]2+
Article Snippet: Paragraph title: Sample Preparation ... The cells were cultivated in RPMI 1640 liquid medium with 20 mM HEPES, stable glutamine (FG 1235, Biochrom AG, Germany), 10% fetal bovine serum (10099-133, Life Technologies, Germany) together with 100 units/mL of penicillin and 100 μ g/mL of streptomycin (15140, Gibco® , Life Technologies GmbH, Germany).

Laser Capture Microdissection:

Article Title: Rad51 Accumulation at Sites of DNA Damage and in Postreplicative Chromatin
Article Snippet: During microirradiation, the cells were kept in RPMI medium containing 25 mM Hepes and 10% FCS (Biochrom). .. Microirradiation was carried out with a laser microdissection system (P.A.L.M.) coupled into a ZEISS Axiovert 100.

Concentration Assay:

Article Title: Overexpression of Inosine 5?-Monophosphate Dehydrogenase Type II Mediates Chemoresistance to Human Osteosarcoma Cells
Article Snippet: Cells were maintained in RPMI 1640 (Lonza GmbH, Wuppertal, Germany) containing 2 mM L-glutamine and 25 mM HEPES and supplemented with fetal calf serum (Biochrom, Berlin, Germany), and 100 U/ml penicillin/streptomycin (Lonza GmbH) at 37°C in a humidified 5% CO2 atmosphere. .. For drug treatment, cisplatin (Sigma-Aldrich, Taufkirchen, Germany) was dissolved in N,N-dimethylformamide at a concentration of 15 mg/ml and added to the culture medium in the indicated concentrations.

Article Title: Molecular Characterization and Functional Analysis of Murine Interleukin 4 Receptor Allotypes
Article Snippet: 15 μg of purified plasmid DNA was electroporated into 107 293-EBNA or TF-1 cells in 0.8 ml of medium (Click's RPMI 1640 [Life Technologies, Eggenstein, Germany], 2 mM l -glutamine, 10 mM Hepes, 100 μg/ml penicillin, 60 ng/ml streptomycin, 13 mM NaHCO3 and 5 × 10−5 M 2-ME) supplemented with 10% (vol/vol) FCS (Biochrom, Berlin, Germany) at 900 μF and 260 V in an Easyject electroporation unit (Eurogentec, Seraing, Belgium). .. The selection of sIL-4R–transfected 293-EBNA cells was started after 36 h by addition of hygromycin (Calbiochem, Bad Soden, Germany) to a final concentration of 0.3 mg/ml.

Article Title: The Mechanism behind Bacterial Lipoprotein Release: Phenol-Soluble Modulins Mediate Toll-Like Receptor 2 Activation via Extracellular Vesicle Release from Staphylococcus aureus
Article Snippet: .. These cells were grown in RPMI medium (Biochrom) supplemented with 10% FCS (Sigma-Aldrich), 20 mM HEPES (Biochrom), penicillin (100 units/ml), streptomycin (100 µg/ml) (Gibco), 1× GlutaMAX (Gibco), and G418 (Biochrom) at a final concentration of 1 mg/ml. .. Calcium fluxes were analyzed by stimulating cells loaded with Fluo-3-AM (Molecular Probes), and the fluorescence was monitored with a FACSCalibur flow cytometer (Becton, Dickinson), as recently described ( ).

Staining:

Article Title: Early Production of the Neutrophil-Derived Lipid Mediators LTB4 and LXA4 Is Modulated by Intracellular Infection with Leishmania major
Article Snippet: After isolation neutrophils were resuspended in complete medium (RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum) and 50 μ M β -mercaptoethanol (all from Sigma-Aldrich, Steinheim, Germany), 4 mM L-glutamine, and 10 mM HEPES (both Biochrom, Berlin, Germany). .. The obtained cell preparations contained > 99% granulocytes according to morphological examination of cytocentrifuge slides stained with Diff Quik (Medion Diagnostics, Düdingen, Switzerland).

Gradient Centrifugation:

Article Title: Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids
Article Snippet: Human PBMCs were prepared from heparinized blood by Ficoll density gradient centrifugation (Ficoll-Paque 1.077 g/ml, GE Healthcare, Freiburg, Germany). .. PHA-stimulated PBMCs (PHA lymphoblasts) were prepared by cultivating human PBMCs for 7 days in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 100 units of penicillin/ml, 0.1 mg streptomycin/ml, 10 mM HEPES and 0.5 μ g/ml PHA (Biochrom, Berlin, Germany).

Article Title: Early Production of the Neutrophil-Derived Lipid Mediators LTB4 and LXA4 Is Modulated by Intracellular Infection with Leishmania major
Article Snippet: Using layered lymphocyte separation medium 1077 (PAA, Pasching, Austria) and Histopaque 1119 (Sigma-Aldrich, Deisenhofen, Germany) first, the separated granulocytes were purified in a discontinuous Percoll (Amersham Biosciences, Uppsala, Sweden) gradient centrifugation. .. After isolation neutrophils were resuspended in complete medium (RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum) and 50 μ M β -mercaptoethanol (all from Sigma-Aldrich, Steinheim, Germany), 4 mM L-glutamine, and 10 mM HEPES (both Biochrom, Berlin, Germany).

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    Biochrom hepes buffered
    Neutrophil extracellular traps are induced by phorbol myristate acetate (PMA) and immobilized immune complex (iIC) under N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid <t>(HEPES)-</t> and bicarbonate-buffered conditions at pH 7.4 . A total of 10 6 neutrophils/ml were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4 and were then stimulated with PMA, iIC, or left untreated. Release of neutrophil extracellular traps (NETs) was monitored for 4 h (PMA) or 7 h (iIC) at 37°C (under 10% CO 2 for bicarbonate-buffered system). (A) Representative real-time kinetics of <t>NET</t> release measured by staining with SYTOXgreen and (B) area under the curve (AUC) values (mean ± SEM) of NET-dependent relative fluorescence intensities (RFUs) as measured by the SYTOXgreen assay. n = 3–9, ** p
    Hepes Buffered, supplied by Biochrom, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Neutrophil extracellular traps are induced by phorbol myristate acetate (PMA) and immobilized immune complex (iIC) under N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)- and bicarbonate-buffered conditions at pH 7.4 . A total of 10 6 neutrophils/ml were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4 and were then stimulated with PMA, iIC, or left untreated. Release of neutrophil extracellular traps (NETs) was monitored for 4 h (PMA) or 7 h (iIC) at 37°C (under 10% CO 2 for bicarbonate-buffered system). (A) Representative real-time kinetics of NET release measured by staining with SYTOXgreen and (B) area under the curve (AUC) values (mean ± SEM) of NET-dependent relative fluorescence intensities (RFUs) as measured by the SYTOXgreen assay. n = 3–9, ** p

    Journal: Frontiers in Immunology

    Article Title: Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2017.00184

    Figure Lengend Snippet: Neutrophil extracellular traps are induced by phorbol myristate acetate (PMA) and immobilized immune complex (iIC) under N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)- and bicarbonate-buffered conditions at pH 7.4 . A total of 10 6 neutrophils/ml were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4 and were then stimulated with PMA, iIC, or left untreated. Release of neutrophil extracellular traps (NETs) was monitored for 4 h (PMA) or 7 h (iIC) at 37°C (under 10% CO 2 for bicarbonate-buffered system). (A) Representative real-time kinetics of NET release measured by staining with SYTOXgreen and (B) area under the curve (AUC) values (mean ± SEM) of NET-dependent relative fluorescence intensities (RFUs) as measured by the SYTOXgreen assay. n = 3–9, ** p

    Article Snippet: NET Formation Is Inducible under Both Bicarbonate- and HEPES-Buffered Conditions As shown in Figures A–C, both PMA and iIC significantly induced the release of NETs by human primary neutrophils at pH 7.4 under both bicarbonate- and HEPES-buffered conditions as detected by SYTOXgreen real-time kinetics and by MPO–DNA (NET) ELISA.

    Techniques: Staining, Fluorescence

    Extracellular acidosis inhibits the release of phorbol myristate acetate (PMA)- and immobilized immune complex (iIC)-induced neutrophil extracellular trap (NETs) under N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid HEPES- and bicarbonate-buffered conditions . 10 6 neutrophils per milliliter were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4, 6.5, 6.0, and 5.5 and were then stimulated with PMA, iIC, or left untreated. Release of NETs was monitored for 4 h (PMA) or 7 h (iIC) by using the SYTOXgreen assay. (A,B,D,E) Representative real-time kinetics and (C,F) area under the curve (AUC) values (mean ± SEM) of NET-dependent relative fluorescence intensities (RFUs) as measured by the SYTOXgreen assay for (A–C) HEPES and (D–E) bicarbonate-buffered conditions. n = 3–9, ** p

    Journal: Frontiers in Immunology

    Article Title: Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2017.00184

    Figure Lengend Snippet: Extracellular acidosis inhibits the release of phorbol myristate acetate (PMA)- and immobilized immune complex (iIC)-induced neutrophil extracellular trap (NETs) under N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid HEPES- and bicarbonate-buffered conditions . 10 6 neutrophils per milliliter were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4, 6.5, 6.0, and 5.5 and were then stimulated with PMA, iIC, or left untreated. Release of NETs was monitored for 4 h (PMA) or 7 h (iIC) by using the SYTOXgreen assay. (A,B,D,E) Representative real-time kinetics and (C,F) area under the curve (AUC) values (mean ± SEM) of NET-dependent relative fluorescence intensities (RFUs) as measured by the SYTOXgreen assay for (A–C) HEPES and (D–E) bicarbonate-buffered conditions. n = 3–9, ** p

    Article Snippet: NET Formation Is Inducible under Both Bicarbonate- and HEPES-Buffered Conditions As shown in Figures A–C, both PMA and iIC significantly induced the release of NETs by human primary neutrophils at pH 7.4 under both bicarbonate- and HEPES-buffered conditions as detected by SYTOXgreen real-time kinetics and by MPO–DNA (NET) ELISA.

    Techniques: Fluorescence

    Effect of extracellular acidosis on neutrophil survival and phagocytosis . (A–D) Neutrophils (10 6 cells/ml) were incubated for 4 or 7 h in (A,B) bicarbonate (10% CO 2 ) or (C,D) N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)-buffered RPMI 1640 at pH 7.4, 6.5, 6.0, and 5.5. Apoptosis and necrosis rate of neutrophils was then analyzed by flow cytometry using Annexin V-FITC and propidium iodide staining. Mean values ± SEM (% cells) from three independent experiments for each pH value are given. * p

    Journal: Frontiers in Immunology

    Article Title: Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2017.00184

    Figure Lengend Snippet: Effect of extracellular acidosis on neutrophil survival and phagocytosis . (A–D) Neutrophils (10 6 cells/ml) were incubated for 4 or 7 h in (A,B) bicarbonate (10% CO 2 ) or (C,D) N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)-buffered RPMI 1640 at pH 7.4, 6.5, 6.0, and 5.5. Apoptosis and necrosis rate of neutrophils was then analyzed by flow cytometry using Annexin V-FITC and propidium iodide staining. Mean values ± SEM (% cells) from three independent experiments for each pH value are given. * p

    Article Snippet: Media Acidification and Cell Culture In this study, HEPES-buffered (Biochrom, Germany) or bicarbonate-buffered RPMI 1640 medium (Sigma-Aldrich, Germany) supplemented with 0.5% human serum albumin (HSA, Baxter, Germany) and 4 mM l -glutamin (Biochrom) was used.

    Techniques: Incubation, Flow Cytometry, Cytometry, Staining

    Extracellular acidosis leads to decreased phorbol myristate acetate (PMA)- and immobilized immune complex (iIC)-induced reactive oxygen species (ROS) production in N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)- and bicarbonate-buffered media . Neutrophils (2 × 10 6 /ml) were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4, 6.5, 6.0, and 5.5 and were then stimulated with PMA, iIC, or left untreated. Real-time analysis of intra- and extracellular myeloperoxidase-dependent ROS was monitored by using the luminol-assay for 1 h at 37°C (under CO 2 for bicarbonate-buffered conditions). (A,B,D,E) Representative real-time kinetics and (C,F) area under the curve (AUC) values (mean ± SEM) of ROS-dependent chemiluminescence intensities (RLUs). n = 3–9 independent experiments (* p

    Journal: Frontiers in Immunology

    Article Title: Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2017.00184

    Figure Lengend Snippet: Extracellular acidosis leads to decreased phorbol myristate acetate (PMA)- and immobilized immune complex (iIC)-induced reactive oxygen species (ROS) production in N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)- and bicarbonate-buffered media . Neutrophils (2 × 10 6 /ml) were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4, 6.5, 6.0, and 5.5 and were then stimulated with PMA, iIC, or left untreated. Real-time analysis of intra- and extracellular myeloperoxidase-dependent ROS was monitored by using the luminol-assay for 1 h at 37°C (under CO 2 for bicarbonate-buffered conditions). (A,B,D,E) Representative real-time kinetics and (C,F) area under the curve (AUC) values (mean ± SEM) of ROS-dependent chemiluminescence intensities (RLUs). n = 3–9 independent experiments (* p

    Article Snippet: Blocking of NHE-1 with DMA significantly reduced the ROS production (Figures A,B) and the NET release (Figures C,D) of PMA- and iIC-stimulated neutrophils under both bicarbonate- and HEPES-buffered conditions.

    Techniques:

    Extracellular acidosis inhibits the release of neutrophil extracellular trap (NET) fibers . Neutrophils (10 6 /ml) were allowed to settle on coverslips for 30 min under bicarbonate- or N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)-buffered conditions at pH 7.4, 6.5, 6.0, and 5.5 and were then stimulated with phorbol myristate acetate (PMA), immobilized immune complex (iIC), or left untreated. PMA-stimulated samples were fixed after 4 h and iIC-stimulated and unstimulated neutrophils after 7 h incubation time. Representative scanning electron images of PMA- and iIC-induced NETs under (A) HEPES and (B) bicarbonate-buffered conditions are shown. The white scale bar represents 20 µm and the black scale bar 10 µm.

    Journal: Frontiers in Immunology

    Article Title: Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2017.00184

    Figure Lengend Snippet: Extracellular acidosis inhibits the release of neutrophil extracellular trap (NET) fibers . Neutrophils (10 6 /ml) were allowed to settle on coverslips for 30 min under bicarbonate- or N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)-buffered conditions at pH 7.4, 6.5, 6.0, and 5.5 and were then stimulated with phorbol myristate acetate (PMA), immobilized immune complex (iIC), or left untreated. PMA-stimulated samples were fixed after 4 h and iIC-stimulated and unstimulated neutrophils after 7 h incubation time. Representative scanning electron images of PMA- and iIC-induced NETs under (A) HEPES and (B) bicarbonate-buffered conditions are shown. The white scale bar represents 20 µm and the black scale bar 10 µm.

    Article Snippet: Blocking of NHE-1 with DMA significantly reduced the ROS production (Figures A,B) and the NET release (Figures C,D) of PMA- and iIC-stimulated neutrophils under both bicarbonate- and HEPES-buffered conditions.

    Techniques: Incubation

    Extracellular acidosis leads to decreased phorbol myristate acetate (PMA)- and immobilized immune complex (iIC)-induced superoxide production in N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)- and bicarbonate-buffered media . Neutrophils (2 × 10 6 /ml) were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4, 6.5, 6.0, and 5.5 and were then stimulated with PMA, iIC, or left untreated. (A–F) Real-time analysis of extracellular superoxide was monitored by using the lucigenin assay for 1 h at 37°C (under CO 2 for bicarbonate-buffered conditions). (A,B,D,E) Show representative real-time kinetics and (C,F) area under the curve (AUC) values (mean ± SEM) of reactive oxygen species (ROS)-dependent chemiluminescence intensities (RLU). n = 6 independent experiments. (G,H) Intracellular ROS production was analyzed by flow cytometry using DHR-123. Cells were preincubated for 30 min in bicarbonate- or HEPES-buffered medium and then stimulated for 30 min with PMA or iIC in the presence of DHR. Mean ± SEM ( n = 3) values of detected rhodamin fluorescence are shown (* p

    Journal: Frontiers in Immunology

    Article Title: Extracellular Acidification Inhibits the ROS-Dependent Formation of Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2017.00184

    Figure Lengend Snippet: Extracellular acidosis leads to decreased phorbol myristate acetate (PMA)- and immobilized immune complex (iIC)-induced superoxide production in N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic-acid (HEPES)- and bicarbonate-buffered media . Neutrophils (2 × 10 6 /ml) were preincubated for 30 min under bicarbonate- or HEPES-buffered conditions at pH 7.4, 6.5, 6.0, and 5.5 and were then stimulated with PMA, iIC, or left untreated. (A–F) Real-time analysis of extracellular superoxide was monitored by using the lucigenin assay for 1 h at 37°C (under CO 2 for bicarbonate-buffered conditions). (A,B,D,E) Show representative real-time kinetics and (C,F) area under the curve (AUC) values (mean ± SEM) of reactive oxygen species (ROS)-dependent chemiluminescence intensities (RLU). n = 6 independent experiments. (G,H) Intracellular ROS production was analyzed by flow cytometry using DHR-123. Cells were preincubated for 30 min in bicarbonate- or HEPES-buffered medium and then stimulated for 30 min with PMA or iIC in the presence of DHR. Mean ± SEM ( n = 3) values of detected rhodamin fluorescence are shown (* p

    Article Snippet: Blocking of NHE-1 with DMA significantly reduced the ROS production (Figures A,B) and the NET release (Figures C,D) of PMA- and iIC-stimulated neutrophils under both bicarbonate- and HEPES-buffered conditions.

    Techniques: Flow Cytometry, Cytometry, Fluorescence