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Carl Roth GmbH ethylenediaminetetraacetic acid
Ethylenediaminetetraacetic Acid, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Clone Assay:

Article Title: Distinct Signaling Cascades of TREM-1, TLR and NLR in Neutrophils and Monocytic Cells
Article Snippet: CHAPS, DTT, EDTA, NaCl, SDS, Tris, thiourea and urea were from Carl Roth (Karlsruhe, Germany), complete protease inhibitor cocktail from Roche (Basel, Switzerland) and NaF, Na-orthovanadate and PMSF from Sigma-Aldrich (Taufkirchen, Germany). .. F(ab')2 fragments of clones 6B1 and 4C9 were prepared using a commercial kit according to the manufacturer's instructions (Pierce, Thermo Scientific, Rockford, Ill., USA).

Centrifugation:

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects
Article Snippet: Mitochondria were solubilized in buffer (1% digitonin [Merck, 300410], 20 mM Tris-HCl, pH 7.4 [Roth, 9090.1], 0.1 mM EDTA, 50 mM NaCl [Roth, P029.5], 10% (w/v) glycerol [Roth, 7530.1], 1 mM PMSF, 10 mM NEM) to a final concentration of 1 µg/µL for 30 min at 4°C. .. Lysates were cleared by centrifugation at 14,000 g for 10 min at 4°C and 10X loading dye was added (5% Coomassie brilliant blue G-250 [Roth, 9598.2], 500 mM 6-aminohexanoic acid [Sigma-Aldrich, 07260], 100 mM Bis-Tris, pH 7.0).

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects
Article Snippet: Cells were harvested in phosphate-buffered saline (PBS; 10 mM Na2 HPO4 [Roth, T876.1], 1.8 mM KH2 PO4 [Roth, 3904.2], 137 mM NaCl [Roth, P029.5], 2.7 mM KCl [Roth, P017.2]), and washed using isolation buffer (300 mM trehalose [Roth, 5151.3], 10 mM KCl [Roth, P017.2],; 10 mM HEPES [Roth, 9105.2], pH 7.4, 1 mM EDTA [Roth, 8040.1], 0.5 mM phenylmethylsulfonylfluoride [PMSF; Roth, 6367.1] and 10 mM N-ethylmaleimide [NEM; Sigma-Aldrich, 128287]). .. Cell debris was removed by centrifugation at 800 g for 10 min. Where required, a sample of the lysed cells was kept for analysis.

Article Title: ?-Fucosidase as a novel convenient biomarker for cellular senescence
Article Snippet: After centrifugation at 14,000 g and 4°C for 5 min, the supernatant was isolated, and the protein content was quantified using a BCA assay kit (Thermo Fischer Scientific). .. To measure hydrolase activities, 5 µL of the supernatant were incubated in 45 µL of hydrolase-specific reaction buffer containing hydrolase-specific 4-MU-glycosides for 1 h at 37°C. α-L-fucosidase was measured in 0.2 M sodium citrate pH 4.5, using 1 mM 4-MU-α-L-fucoside (Sigma), β-galactosidase in 66 mM NaCl, 66 mM Na2 HPO4 , 33 mM sodium citrate, pH 4.6, using 1 mM 4-MU-β-D-galactoside (Roth) as described. β-glucuronidase was measured in 200 mM sodium acetate buffer, pH 5.0, 10 mM EDTA, 0.01% BSA, 0.1% Triton X-100 using 2.5 mM 4-MU-β-D-glucuronide (Roth) according to Sperker et al. Enzymatic reactions were stopped by the addition of 0.2 mL Na2 CO3 , and reaction mixtures were centrifuged for 10 min at 2,000 g. The cleared supernatant was used to determine the concentration of 4-MU by fluorescence detection (excitation 355 nm, emission 460 nm) using an Infinite 200 Reader (Tecan, Männedorf, Switzerland).

Article Title: A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells
Article Snippet: The standard protocol included 1:3 dilution of whole blood with phosphate buffered saline (PBS) and centrifugation at 1,000 x g for 20 min without brakes using Biocoll (1.077 g/ml, Biochrom GmbH, Berlin, Germany). .. After removing the supernatant above the leukocyte layer, the latter was carefully collected with a plastic pipette and washed with washing buffer (PBS with 0.5% bovine serum albumin (Sigma-Aldrich, Steinheim, Germany) and 2 mM ethylenediaminetetraacetic acid (Carl Roth GmbH & Co. KG, Karlsruhe, Germany)) at 300 x g for 10 min (brakes: level two).

Electrophoresis:

Article Title: Selection of Shine-Dalgarno sequences in plastids
Article Snippet: Protein extraction and immunoblot analyses Plant total soluble protein (TSP) was extracted from leaf samples homogenized in a buffer containing 50 mM HEPES, 10 mM KAc, 5 mM MgAc, 1 mM EDTA, 1 mM Pefablock (Roth, Karlsruhe, Germany) and 1 mM DTT (pH 7.5). .. Extracted protein samples were separated by electrophoresis in 15–20% SDS–polyacrylamide gradient gels and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare).

Ex Vivo:

Article Title: Protection of Batf3‐deficient mice from experimental cerebral malaria correlates with impaired cytotoxic T‐cell responses and immune regulation
Article Snippet: Paragraph title: Preparation of organs and lymphocytes for ex vivo analysis ... The interphase containing the lymphocytes was carefully transferred into a new tube and washed with 1 × PBS/1% FCS (PAA, Cölbe, Germany)/2 mm EDTA (Roth, Karlsruhe, Germany).

Incubation:

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects
Article Snippet: Mitochondria were solubilized in buffer (1% digitonin [Merck, 300410], 20 mM Tris-HCl, pH 7.4 [Roth, 9090.1], 0.1 mM EDTA, 50 mM NaCl [Roth, P029.5], 10% (w/v) glycerol [Roth, 7530.1], 1 mM PMSF, 10 mM NEM) to a final concentration of 1 µg/µL for 30 min at 4°C. .. For subsequent 2D analysis, the appropriate lanes were excised from the BN-PAGE gel and incubated in equilibration buffer (25 mM Tris, pH 8.5, 0.2 M glycine, 1% SDS [Roth, 5136.1] and 2% 1,4-dithiothreitol [Roth, 6908.1]) for 10 min prior to further separation by SDS-PAGE.

Article Title: Distinct Clones of Yersinia pestis Caused the Black Death
Article Snippet: .. Aliquots of 0.38–0.5 g powder were incubated overnight on a rotary mixer at 37°C in a decalcification and digestion solution consisting of 0.5 M EDTA (pH 8; Roth, Karlsruhe, Germany), 0.4% N-Laurylsarcosine and 0.46 mg/ml Proteinase K (Roche, Germany). .. Post-digestion, DNA was extracted using phenol/chloroform/isoamyl alcohol (25∶24∶1 Roth, Karlsruhe, Germany) and trichlormethan/chloroform (ROTIPURAN ≥99%, p.a.

Article Title: ?-Fucosidase as a novel convenient biomarker for cellular senescence
Article Snippet: .. To measure hydrolase activities, 5 µL of the supernatant were incubated in 45 µL of hydrolase-specific reaction buffer containing hydrolase-specific 4-MU-glycosides for 1 h at 37°C. α-L-fucosidase was measured in 0.2 M sodium citrate pH 4.5, using 1 mM 4-MU-α-L-fucoside (Sigma), β-galactosidase in 66 mM NaCl, 66 mM Na2 HPO4 , 33 mM sodium citrate, pH 4.6, using 1 mM 4-MU-β-D-galactoside (Roth) as described. β-glucuronidase was measured in 200 mM sodium acetate buffer, pH 5.0, 10 mM EDTA, 0.01% BSA, 0.1% Triton X-100 using 2.5 mM 4-MU-β-D-glucuronide (Roth) according to Sperker et al. Enzymatic reactions were stopped by the addition of 0.2 mL Na2 CO3 , and reaction mixtures were centrifuged for 10 min at 2,000 g. The cleared supernatant was used to determine the concentration of 4-MU by fluorescence detection (excitation 355 nm, emission 460 nm) using an Infinite 200 Reader (Tecan, Männedorf, Switzerland). ..

Article Title: Characterization of MtfA, a Novel Regulatory Output Signal Protein of the Glucose-Phosphotransferase System in Escherichia coli K-12
Article Snippet: The samples were then filled to a final volume of 1 ml with Tris-buffer (pH 8.0) and incubated at 37°C for 4 h. The absorbance was measured against a blank sample without protein (500 μl of l -alanine 4-nitroanilide with 500 μl of Tris buffer) at 390 nm. .. If indicated, we added the inhibitors AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride; Applichem, Darmstadt, Germany], EDTA (Roth, Karlsruhe, Germany), or phenanthroline (Sigma) in the denoted concentrations to the assay.

Article Title: Viscoelastic properties of vimentin originate from nonequilibrium conformational changes
Article Snippet: .. The pellet was transferred to a cooled douncer, homogenized with 16 ml of tris buffer (50 mM; pH 8.0; Carl Roth GmbH) containing saccharose (25%, w/v), EDTA (1 mM; Carl Roth GmbH), and lysozyme (10 mg/ml; Roche Diagnostics), and incubated on ice for 30 min. .. Eight hundred microliters of MgCl2 solution (1 M; Sigma-Aldrich), 80 μl of deoxyribonuclease I (DNase 1; 50 mg/ml, Sigma-Aldrich) in tris buffer (10 mM; pH 7.5) containing NaCl (100 mM; Carl Roth GmbH), 80 μl of ribonuclease A (RNase A; 10 mg/ml; Roche Diagnostics) in tris buffer (100 mM; pH 7.5), 800 μl of saturated phenylmethylsulfonyl fluoride (PMSF; Serva) in ethanol, and 1.6 ml of 10% NP-40 (Roche Diagnostics) were added and mixed several times by homogenization.

Article Title: Protection of Batf3‐deficient mice from experimental cerebral malaria correlates with impaired cytotoxic T‐cell responses and immune regulation
Article Snippet: The interphase containing the lymphocytes was carefully transferred into a new tube and washed with 1 × PBS/1% FCS (PAA, Cölbe, Germany)/2 mm EDTA (Roth, Karlsruhe, Germany). .. Then, single cells were resuspended in RPMI medium (Sigma‐Aldrich, Munich, Germany) containing 10% fetal calf serum (PAA Laboratories GmbH, Cölbe, Germany), 1% penicillin/streptomycin (Lonza, Wuppertal, Germany), 1% l ‐glutamine (PAA), and used for either flow cytometric staining or in vitro incubation.

Activity Assay:

Article Title: ?-Fucosidase as a novel convenient biomarker for cellular senescence
Article Snippet: To measure hydrolase activities, 5 µL of the supernatant were incubated in 45 µL of hydrolase-specific reaction buffer containing hydrolase-specific 4-MU-glycosides for 1 h at 37°C. α-L-fucosidase was measured in 0.2 M sodium citrate pH 4.5, using 1 mM 4-MU-α-L-fucoside (Sigma), β-galactosidase in 66 mM NaCl, 66 mM Na2 HPO4 , 33 mM sodium citrate, pH 4.6, using 1 mM 4-MU-β-D-galactoside (Roth) as described. β-glucuronidase was measured in 200 mM sodium acetate buffer, pH 5.0, 10 mM EDTA, 0.01% BSA, 0.1% Triton X-100 using 2.5 mM 4-MU-β-D-glucuronide (Roth) according to Sperker et al. Enzymatic reactions were stopped by the addition of 0.2 mL Na2 CO3 , and reaction mixtures were centrifuged for 10 min at 2,000 g. The cleared supernatant was used to determine the concentration of 4-MU by fluorescence detection (excitation 355 nm, emission 460 nm) using an Infinite 200 Reader (Tecan, Männedorf, Switzerland). .. Relative enzyme activities are given as fold control, whereas 1-fold was the average absolute enzyme activity of all three control samples harvested on days 3, 5 and 7 of senescence induction experiments.

Article Title: Characterization of MtfA, a Novel Regulatory Output Signal Protein of the Glucose-Phosphotransferase System in Escherichia coli K-12
Article Snippet: Paragraph title: Protease activity assays. ... If indicated, we added the inhibitors AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride; Applichem, Darmstadt, Germany], EDTA (Roth, Karlsruhe, Germany), or phenanthroline (Sigma) in the denoted concentrations to the assay.

Article Title: Polymorphic variation in TPMT is the principal determinant of TPMT phenotype: a meta-analysis of three genome-wide association studies
Article Snippet: Briefly, approximately 1 g of tissue was homogenized in 1 mM EDTA, 1 mM DTT, 10 mM HEPES pH7.4, 0.2 mM Pefabloc (Roth, Karlsruhe, Germany) and 0.15 mM KCl and differentially centrifuged at 15 000 g and 105 000 g. The final cytosolic supernatant was immediately frozen in liquid nitrogen in aliquots and stored at −80°C until use. .. Cytosol used for TPMT activity measurements was available for liver samples of 124 patients (n=124; 54 males, 70 females; age: median 58 years, range 7 to 85 years).

Article Title: Quantitative analysis of the human T cell palmitome
Article Snippet: Cell pellets were lysed for 30 minutes on ice in ABE lysis buffer (50 mM Tris (pH 7.4) (Roth), 150 mM NaCl (Roth), 10 mM MgCl2 (AppliChem), 10 mM KCl (Sigma-Aldrich), 500 μM EDTA (Roth), 100 μM Na3 VO4 (Sigma-Aldrich), 20 mM N-ethylmaleimide (NEM) (Thermo), 1 mM PMSF, 1.7% Triton X-100 (Roth), 1 mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich), 100 μM methyl arachidonyl fluorophosphonate (MAFP) (Sigma-Aldrich), cOmplete EDTA-free protease inhibitor cocktail (PI-cocktail) (Roche) at a protein concentration of 3 mg/ml, then spun at 16,100 × g, 4 °C, 10 min to remove cell debris. .. The inclusion of MAFP, a lipase inhibitor, ensures the palmitoylation state is arrested by inhibiting all palmitoyl protein thioesterase activity.

Expressing:

Article Title: Viscoelastic properties of vimentin originate from nonequilibrium conformational changes
Article Snippet: Paragraph title: Vimentin expression and purification protocol ... The pellet was transferred to a cooled douncer, homogenized with 16 ml of tris buffer (50 mM; pH 8.0; Carl Roth GmbH) containing saccharose (25%, w/v), EDTA (1 mM; Carl Roth GmbH), and lysozyme (10 mg/ml; Roche Diagnostics), and incubated on ice for 30 min.

BIA-KA:

Article Title: ?-Fucosidase as a novel convenient biomarker for cellular senescence
Article Snippet: After centrifugation at 14,000 g and 4°C for 5 min, the supernatant was isolated, and the protein content was quantified using a BCA assay kit (Thermo Fischer Scientific). .. To measure hydrolase activities, 5 µL of the supernatant were incubated in 45 µL of hydrolase-specific reaction buffer containing hydrolase-specific 4-MU-glycosides for 1 h at 37°C. α-L-fucosidase was measured in 0.2 M sodium citrate pH 4.5, using 1 mM 4-MU-α-L-fucoside (Sigma), β-galactosidase in 66 mM NaCl, 66 mM Na2 HPO4 , 33 mM sodium citrate, pH 4.6, using 1 mM 4-MU-β-D-galactoside (Roth) as described. β-glucuronidase was measured in 200 mM sodium acetate buffer, pH 5.0, 10 mM EDTA, 0.01% BSA, 0.1% Triton X-100 using 2.5 mM 4-MU-β-D-glucuronide (Roth) according to Sperker et al. Enzymatic reactions were stopped by the addition of 0.2 mL Na2 CO3 , and reaction mixtures were centrifuged for 10 min at 2,000 g. The cleared supernatant was used to determine the concentration of 4-MU by fluorescence detection (excitation 355 nm, emission 460 nm) using an Infinite 200 Reader (Tecan, Männedorf, Switzerland).

Transplantation Assay:

Article Title: Polymorphic variation in TPMT is the principal determinant of TPMT phenotype: a meta-analysis of three genome-wide association studies
Article Snippet: Histologically normal human liver tissues as well as corresponding blood samples were collected from patients undergoing liver surgery at the Department of General, Visceral and Transplantation Surgery (University Medical Center Charité, Berlin, Germany) as previously described. .. Briefly, approximately 1 g of tissue was homogenized in 1 mM EDTA, 1 mM DTT, 10 mM HEPES pH7.4, 0.2 mM Pefabloc (Roth, Karlsruhe, Germany) and 0.15 mM KCl and differentially centrifuged at 15 000 g and 105 000 g. The final cytosolic supernatant was immediately frozen in liquid nitrogen in aliquots and stored at −80°C until use.

Western Blot:

Article Title: Distinct Signaling Cascades of TREM-1, TLR and NLR in Neutrophils and Monocytic Cells
Article Snippet: CHAPS, DTT, EDTA, NaCl, SDS, Tris, thiourea and urea were from Carl Roth (Karlsruhe, Germany), complete protease inhibitor cocktail from Roche (Basel, Switzerland) and NaF, Na-orthovanadate and PMSF from Sigma-Aldrich (Taufkirchen, Germany). .. Antibodies against Akt (clone C67E7), p-Akt (clone 193H12), p38MAPK, p-p38MAPK (clone 3D7) and HRP-linked secondary antibodies for Western blot analyses were from Cell Signaling (Danvers, Mass., USA).

High Performance Liquid Chromatography:

Article Title: Verticillium longisporum infection induces organ-specific glucosinolate degradation in Arabidopsis thaliana
Article Snippet: .. Chemicals 2Prop-ITC, (≥99%), benzonitrile (≥99.9%), 3-butenenitrile (2Prop-CN, ≥98%), 4-pentenenitrile (3But-CN, ≥97%), 3-phenylpropanenitrile (2PE-CN, ≥99%) and sucrose-sodium nitrate media were purchased from Sigma-Aldrich Chemie GmbH, Steinheim, Germany; IAN, (≥98%) from Acros Organics (Fischer Scientific GmbH, Schwerte, Germany); 3-butenyl ITC (3But-ITC, ≥95%) and 4-pentenyl ITC (4Pent-ITC, ≥95%) were purchased from TCI Deutschland GmbH, Eschborn, Germany; 3-hydroxypropionitrile was purchased from Thermo Fischer Scientific, Erembodegem, Belgium; 4-(methylthio)butyl ITC (4MTB-ITC, ≥98%) was purchased from Santa Cruz Biotechnology, Heidelberg, Germany; 5-(methylsulfinyl)pentyl ITC (5MSOP-ITC) was purchased from Enzo Life Sciences GmbH, Lörrach, Germany; 1-cyano-2,3-epithiopropane (CETP), (≥95%) was purchased from Taros Chemicals GmbH Co. KG, Dortmund, Germany; 4-hydroxybenzyl GLS (≥97%), methylene chloride (GC Ultra Grade), Tris, EDTA, NaCl, CTAB, chloroform/isoamylalcohol (24:1), β-mercaptoethanol and phenol/chloroform/isoamylalcohol (25:24:1) from Carl Roth GmbH, Karlsruhe, Germany; acetonitrile (Ultra Gradient HPLC grade) was purchased from J.T. .. Baker, Deventer, The Netherlands and NaSO4 (≥99%) and methanol ( > 99.9) were purchased from VWR International GmbH, Darmstadt, Germany.

Flow Cytometry:

Article Title: A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells
Article Snippet: After removing the supernatant above the leukocyte layer, the latter was carefully collected with a plastic pipette and washed with washing buffer (PBS with 0.5% bovine serum albumin (Sigma-Aldrich, Steinheim, Germany) and 2 mM ethylenediaminetetraacetic acid (Carl Roth GmbH & Co. KG, Karlsruhe, Germany)) at 300 x g for 10 min (brakes: level two). .. The two leukocyte recovery protocols were compared by cell counting and flow cytometric analysis focusing on the forward scatter/ side scatter distribution of the cells.

Article Title: Protection of Batf3‐deficient mice from experimental cerebral malaria correlates with impaired cytotoxic T‐cell responses and immune regulation
Article Snippet: The interphase containing the lymphocytes was carefully transferred into a new tube and washed with 1 × PBS/1% FCS (PAA, Cölbe, Germany)/2 mm EDTA (Roth, Karlsruhe, Germany). .. Then, single cells were resuspended in RPMI medium (Sigma‐Aldrich, Munich, Germany) containing 10% fetal calf serum (PAA Laboratories GmbH, Cölbe, Germany), 1% penicillin/streptomycin (Lonza, Wuppertal, Germany), 1% l ‐glutamine (PAA), and used for either flow cytometric staining or in vitro incubation.

Gas Chromatography-Mass Spectrometry:

Article Title: Verticillium longisporum infection induces organ-specific glucosinolate degradation in Arabidopsis thaliana
Article Snippet: Chemicals 2Prop-ITC, (≥99%), benzonitrile (≥99.9%), 3-butenenitrile (2Prop-CN, ≥98%), 4-pentenenitrile (3But-CN, ≥97%), 3-phenylpropanenitrile (2PE-CN, ≥99%) and sucrose-sodium nitrate media were purchased from Sigma-Aldrich Chemie GmbH, Steinheim, Germany; IAN, (≥98%) from Acros Organics (Fischer Scientific GmbH, Schwerte, Germany); 3-butenyl ITC (3But-ITC, ≥95%) and 4-pentenyl ITC (4Pent-ITC, ≥95%) were purchased from TCI Deutschland GmbH, Eschborn, Germany; 3-hydroxypropionitrile was purchased from Thermo Fischer Scientific, Erembodegem, Belgium; 4-(methylthio)butyl ITC (4MTB-ITC, ≥98%) was purchased from Santa Cruz Biotechnology, Heidelberg, Germany; 5-(methylsulfinyl)pentyl ITC (5MSOP-ITC) was purchased from Enzo Life Sciences GmbH, Lörrach, Germany; 1-cyano-2,3-epithiopropane (CETP), (≥95%) was purchased from Taros Chemicals GmbH Co. KG, Dortmund, Germany; 4-hydroxybenzyl GLS (≥97%), methylene chloride (GC Ultra Grade), Tris, EDTA, NaCl, CTAB, chloroform/isoamylalcohol (24:1), β-mercaptoethanol and phenol/chloroform/isoamylalcohol (25:24:1) from Carl Roth GmbH, Karlsruhe, Germany; acetonitrile (Ultra Gradient HPLC grade) was purchased from J.T. .. All solvents were of LC or GC-MS grade.

Protease Inhibitor:

Article Title: ?-Fucosidase as a novel convenient biomarker for cellular senescence
Article Snippet: The lysis buffer contained 20 mM TRIS-HCl pH 7.4, 0.2% Triton X-100 and complete protease inhibitor cocktail (Roche). .. To measure hydrolase activities, 5 µL of the supernatant were incubated in 45 µL of hydrolase-specific reaction buffer containing hydrolase-specific 4-MU-glycosides for 1 h at 37°C. α-L-fucosidase was measured in 0.2 M sodium citrate pH 4.5, using 1 mM 4-MU-α-L-fucoside (Sigma), β-galactosidase in 66 mM NaCl, 66 mM Na2 HPO4 , 33 mM sodium citrate, pH 4.6, using 1 mM 4-MU-β-D-galactoside (Roth) as described. β-glucuronidase was measured in 200 mM sodium acetate buffer, pH 5.0, 10 mM EDTA, 0.01% BSA, 0.1% Triton X-100 using 2.5 mM 4-MU-β-D-glucuronide (Roth) according to Sperker et al. Enzymatic reactions were stopped by the addition of 0.2 mL Na2 CO3 , and reaction mixtures were centrifuged for 10 min at 2,000 g. The cleared supernatant was used to determine the concentration of 4-MU by fluorescence detection (excitation 355 nm, emission 460 nm) using an Infinite 200 Reader (Tecan, Männedorf, Switzerland).

Article Title: Quantitative analysis of the human T cell palmitome
Article Snippet: .. Cell pellets were lysed for 30 minutes on ice in ABE lysis buffer (50 mM Tris (pH 7.4) (Roth), 150 mM NaCl (Roth), 10 mM MgCl2 (AppliChem), 10 mM KCl (Sigma-Aldrich), 500 μM EDTA (Roth), 100 μM Na3 VO4 (Sigma-Aldrich), 20 mM N-ethylmaleimide (NEM) (Thermo), 1 mM PMSF, 1.7% Triton X-100 (Roth), 1 mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich), 100 μM methyl arachidonyl fluorophosphonate (MAFP) (Sigma-Aldrich), cOmplete EDTA-free protease inhibitor cocktail (PI-cocktail) (Roche) at a protein concentration of 3 mg/ml, then spun at 16,100 × g, 4 °C, 10 min to remove cell debris. ..

Article Title: Distinct Signaling Cascades of TREM-1, TLR and NLR in Neutrophils and Monocytic Cells
Article Snippet: .. CHAPS, DTT, EDTA, NaCl, SDS, Tris, thiourea and urea were from Carl Roth (Karlsruhe, Germany), complete protease inhibitor cocktail from Roche (Basel, Switzerland) and NaF, Na-orthovanadate and PMSF from Sigma-Aldrich (Taufkirchen, Germany). .. Antibodies against Akt (clone C67E7), p-Akt (clone 193H12), p38MAPK, p-p38MAPK (clone 3D7) and HRP-linked secondary antibodies for Western blot analyses were from Cell Signaling (Danvers, Mass., USA).

Transferring:

Article Title: A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells
Article Snippet: .. After removing the supernatant above the leukocyte layer, the latter was carefully collected with a plastic pipette and washed with washing buffer (PBS with 0.5% bovine serum albumin (Sigma-Aldrich, Steinheim, Germany) and 2 mM ethylenediaminetetraacetic acid (Carl Roth GmbH & Co. KG, Karlsruhe, Germany)) at 300 x g for 10 min (brakes: level two). .. The two leukocyte recovery protocols were compared by cell counting and flow cytometric analysis focusing on the forward scatter/ side scatter distribution of the cells.

Generated:

Article Title: ?-Fucosidase as a novel convenient biomarker for cellular senescence
Article Snippet: To this end, senescent cells were generated as described above. .. To measure hydrolase activities, 5 µL of the supernatant were incubated in 45 µL of hydrolase-specific reaction buffer containing hydrolase-specific 4-MU-glycosides for 1 h at 37°C. α-L-fucosidase was measured in 0.2 M sodium citrate pH 4.5, using 1 mM 4-MU-α-L-fucoside (Sigma), β-galactosidase in 66 mM NaCl, 66 mM Na2 HPO4 , 33 mM sodium citrate, pH 4.6, using 1 mM 4-MU-β-D-galactoside (Roth) as described. β-glucuronidase was measured in 200 mM sodium acetate buffer, pH 5.0, 10 mM EDTA, 0.01% BSA, 0.1% Triton X-100 using 2.5 mM 4-MU-β-D-glucuronide (Roth) according to Sperker et al. Enzymatic reactions were stopped by the addition of 0.2 mL Na2 CO3 , and reaction mixtures were centrifuged for 10 min at 2,000 g. The cleared supernatant was used to determine the concentration of 4-MU by fluorescence detection (excitation 355 nm, emission 460 nm) using an Infinite 200 Reader (Tecan, Männedorf, Switzerland).

Article Title: Protection of Batf3‐deficient mice from experimental cerebral malaria correlates with impaired cytotoxic T‐cell responses and immune regulation
Article Snippet: After extraction, organs were cut into small pieces and digested with 0·5 mg/ml collagenase A (Roche, Basel, Switzerland), at 37° for 30 min. After that, 10 ml washing buffer (1 × PBS 1% FCS 2 mm EDTA) were added and a single‐cell suspension was generated by homogenizing the tissue with the help of a fine metal sieve and washed with 1 × PBS. .. The interphase containing the lymphocytes was carefully transferred into a new tube and washed with 1 × PBS/1% FCS (PAA, Cölbe, Germany)/2 mm EDTA (Roth, Karlsruhe, Germany).

other:

Article Title: Acoustic radiation force impulse imaging of kidneys – a phantom study
Article Snippet: Components used for phantom manufacturing included: distilled water, gelatin (Polskie Odczynniki Chemiczne S.A., Poland), 5 m% n-propanol (Polskie Odczynniki Chemiczne S.A., Poland), 5 m% graphite flakes median size 7–10 micron (Alfa Aesar, Germany), 0.2 m% glutaral aldehyde (Merck-Schuchardt, Germany) and 0.2 m% EDTA (Carl Roth, Germany).

Protein Concentration:

Article Title: Quantitative analysis of the human T cell palmitome
Article Snippet: .. Cell pellets were lysed for 30 minutes on ice in ABE lysis buffer (50 mM Tris (pH 7.4) (Roth), 150 mM NaCl (Roth), 10 mM MgCl2 (AppliChem), 10 mM KCl (Sigma-Aldrich), 500 μM EDTA (Roth), 100 μM Na3 VO4 (Sigma-Aldrich), 20 mM N-ethylmaleimide (NEM) (Thermo), 1 mM PMSF, 1.7% Triton X-100 (Roth), 1 mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich), 100 μM methyl arachidonyl fluorophosphonate (MAFP) (Sigma-Aldrich), cOmplete EDTA-free protease inhibitor cocktail (PI-cocktail) (Roche) at a protein concentration of 3 mg/ml, then spun at 16,100 × g, 4 °C, 10 min to remove cell debris. ..

Binding Assay:

Article Title: The effect of endodontic treatment using different intracanal medicaments on periodontal attachment level in concurrent endodontic-periodontal lesions: A randomized controlled trial
Article Snippet: Canals were enlarged three times larger than the initial binding file followed by step back and irrigated with 10 ml NaOCl (1%) using a 30-gauge NaviTip FX (Ultradent Products Inc, South Jordan, UT, USA). .. Smear layer was removed by irrigating with 5.0 mL 17% EDTA (Roth International Ltd, Chicago, IL, USA) for 1 min followed by 5.0 mL 1% NaOCl, and the final irrigant being 5 ml of distilled water.

DNA Extraction:

Article Title: Distinct Clones of Yersinia pestis Caused the Black Death
Article Snippet: Paragraph title: Sample preparation and DNA extraction ... Aliquots of 0.38–0.5 g powder were incubated overnight on a rotary mixer at 37°C in a decalcification and digestion solution consisting of 0.5 M EDTA (pH 8; Roth, Karlsruhe, Germany), 0.4% N-Laurylsarcosine and 0.46 mg/ml Proteinase K (Roche, Germany).

Fluorescence:

Article Title: ?-Fucosidase as a novel convenient biomarker for cellular senescence
Article Snippet: .. To measure hydrolase activities, 5 µL of the supernatant were incubated in 45 µL of hydrolase-specific reaction buffer containing hydrolase-specific 4-MU-glycosides for 1 h at 37°C. α-L-fucosidase was measured in 0.2 M sodium citrate pH 4.5, using 1 mM 4-MU-α-L-fucoside (Sigma), β-galactosidase in 66 mM NaCl, 66 mM Na2 HPO4 , 33 mM sodium citrate, pH 4.6, using 1 mM 4-MU-β-D-galactoside (Roth) as described. β-glucuronidase was measured in 200 mM sodium acetate buffer, pH 5.0, 10 mM EDTA, 0.01% BSA, 0.1% Triton X-100 using 2.5 mM 4-MU-β-D-glucuronide (Roth) according to Sperker et al. Enzymatic reactions were stopped by the addition of 0.2 mL Na2 CO3 , and reaction mixtures were centrifuged for 10 min at 2,000 g. The cleared supernatant was used to determine the concentration of 4-MU by fluorescence detection (excitation 355 nm, emission 460 nm) using an Infinite 200 Reader (Tecan, Männedorf, Switzerland). ..

Isolation:

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects
Article Snippet: .. Cells were harvested in phosphate-buffered saline (PBS; 10 mM Na2 HPO4 [Roth, T876.1], 1.8 mM KH2 PO4 [Roth, 3904.2], 137 mM NaCl [Roth, P029.5], 2.7 mM KCl [Roth, P017.2]), and washed using isolation buffer (300 mM trehalose [Roth, 5151.3], 10 mM KCl [Roth, P017.2],; 10 mM HEPES [Roth, 9105.2], pH 7.4, 1 mM EDTA [Roth, 8040.1], 0.5 mM phenylmethylsulfonylfluoride [PMSF; Roth, 6367.1] and 10 mM N-ethylmaleimide [NEM; Sigma-Aldrich, 128287]). .. Cells in isolation buffer were homogenized on ice using a Potter S homogenizer (Sartorius, 8533024).

Article Title: ?-Fucosidase as a novel convenient biomarker for cellular senescence
Article Snippet: After centrifugation at 14,000 g and 4°C for 5 min, the supernatant was isolated, and the protein content was quantified using a BCA assay kit (Thermo Fischer Scientific). .. To measure hydrolase activities, 5 µL of the supernatant were incubated in 45 µL of hydrolase-specific reaction buffer containing hydrolase-specific 4-MU-glycosides for 1 h at 37°C. α-L-fucosidase was measured in 0.2 M sodium citrate pH 4.5, using 1 mM 4-MU-α-L-fucoside (Sigma), β-galactosidase in 66 mM NaCl, 66 mM Na2 HPO4 , 33 mM sodium citrate, pH 4.6, using 1 mM 4-MU-β-D-galactoside (Roth) as described. β-glucuronidase was measured in 200 mM sodium acetate buffer, pH 5.0, 10 mM EDTA, 0.01% BSA, 0.1% Triton X-100 using 2.5 mM 4-MU-β-D-glucuronide (Roth) according to Sperker et al. Enzymatic reactions were stopped by the addition of 0.2 mL Na2 CO3 , and reaction mixtures were centrifuged for 10 min at 2,000 g. The cleared supernatant was used to determine the concentration of 4-MU by fluorescence detection (excitation 355 nm, emission 460 nm) using an Infinite 200 Reader (Tecan, Männedorf, Switzerland).

Article Title: A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells
Article Snippet: Paragraph title: Leukocyte isolation ... After removing the supernatant above the leukocyte layer, the latter was carefully collected with a plastic pipette and washed with washing buffer (PBS with 0.5% bovine serum albumin (Sigma-Aldrich, Steinheim, Germany) and 2 mM ethylenediaminetetraacetic acid (Carl Roth GmbH & Co. KG, Karlsruhe, Germany)) at 300 x g for 10 min (brakes: level two).

Labeling:

Article Title: Quantitative analysis of the human T cell palmitome
Article Snippet: Cell pellets were lysed for 30 minutes on ice in ABE lysis buffer (50 mM Tris (pH 7.4) (Roth), 150 mM NaCl (Roth), 10 mM MgCl2 (AppliChem), 10 mM KCl (Sigma-Aldrich), 500 μM EDTA (Roth), 100 μM Na3 VO4 (Sigma-Aldrich), 20 mM N-ethylmaleimide (NEM) (Thermo), 1 mM PMSF, 1.7% Triton X-100 (Roth), 1 mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich), 100 μM methyl arachidonyl fluorophosphonate (MAFP) (Sigma-Aldrich), cOmplete EDTA-free protease inhibitor cocktail (PI-cocktail) (Roche) at a protein concentration of 3 mg/ml, then spun at 16,100 × g, 4 °C, 10 min to remove cell debris. .. Chloroform-methanol (CM) precipitation steps were performed between each chemical labeling step to ensure complete removal of previous reagents.

Purification:

Article Title: Polymorphic variation in TPMT is the principal determinant of TPMT phenotype: a meta-analysis of three genome-wide association studies
Article Snippet: Briefly, approximately 1 g of tissue was homogenized in 1 mM EDTA, 1 mM DTT, 10 mM HEPES pH7.4, 0.2 mM Pefabloc (Roth, Karlsruhe, Germany) and 0.15 mM KCl and differentially centrifuged at 15 000 g and 105 000 g. The final cytosolic supernatant was immediately frozen in liquid nitrogen in aliquots and stored at −80°C until use. .. Purification of genomic DNA from EDTA blood samples was performed as described previously, and DNA samples were available for 150 liver samples.

Article Title: Viscoelastic properties of vimentin originate from nonequilibrium conformational changes
Article Snippet: Paragraph title: Vimentin expression and purification protocol ... The pellet was transferred to a cooled douncer, homogenized with 16 ml of tris buffer (50 mM; pH 8.0; Carl Roth GmbH) containing saccharose (25%, w/v), EDTA (1 mM; Carl Roth GmbH), and lysozyme (10 mg/ml; Roche Diagnostics), and incubated on ice for 30 min.

Article Title: Distinct Signaling Cascades of TREM-1, TLR and NLR in Neutrophils and Monocytic Cells
Article Snippet: CHAPS, DTT, EDTA, NaCl, SDS, Tris, thiourea and urea were from Carl Roth (Karlsruhe, Germany), complete protease inhibitor cocktail from Roche (Basel, Switzerland) and NaF, Na-orthovanadate and PMSF from Sigma-Aldrich (Taufkirchen, Germany). .. Anti-TREM-1 (clones 1C5 or 6B1) and mouse IgG1 (clone 4C9) used as isotype control were purified from hybridoma supernatants as described previously [ ].

Protein Extraction:

Article Title: Selection of Shine-Dalgarno sequences in plastids
Article Snippet: .. Protein extraction and immunoblot analyses Plant total soluble protein (TSP) was extracted from leaf samples homogenized in a buffer containing 50 mM HEPES, 10 mM KAc, 5 mM MgAc, 1 mM EDTA, 1 mM Pefablock (Roth, Karlsruhe, Germany) and 1 mM DTT (pH 7.5). ..

Lysis:

Article Title: ?-Fucosidase as a novel convenient biomarker for cellular senescence
Article Snippet: The lysis buffer contained 20 mM TRIS-HCl pH 7.4, 0.2% Triton X-100 and complete protease inhibitor cocktail (Roche). .. To measure hydrolase activities, 5 µL of the supernatant were incubated in 45 µL of hydrolase-specific reaction buffer containing hydrolase-specific 4-MU-glycosides for 1 h at 37°C. α-L-fucosidase was measured in 0.2 M sodium citrate pH 4.5, using 1 mM 4-MU-α-L-fucoside (Sigma), β-galactosidase in 66 mM NaCl, 66 mM Na2 HPO4 , 33 mM sodium citrate, pH 4.6, using 1 mM 4-MU-β-D-galactoside (Roth) as described. β-glucuronidase was measured in 200 mM sodium acetate buffer, pH 5.0, 10 mM EDTA, 0.01% BSA, 0.1% Triton X-100 using 2.5 mM 4-MU-β-D-glucuronide (Roth) according to Sperker et al. Enzymatic reactions were stopped by the addition of 0.2 mL Na2 CO3 , and reaction mixtures were centrifuged for 10 min at 2,000 g. The cleared supernatant was used to determine the concentration of 4-MU by fluorescence detection (excitation 355 nm, emission 460 nm) using an Infinite 200 Reader (Tecan, Männedorf, Switzerland).

Article Title: Quantitative analysis of the human T cell palmitome
Article Snippet: .. Cell pellets were lysed for 30 minutes on ice in ABE lysis buffer (50 mM Tris (pH 7.4) (Roth), 150 mM NaCl (Roth), 10 mM MgCl2 (AppliChem), 10 mM KCl (Sigma-Aldrich), 500 μM EDTA (Roth), 100 μM Na3 VO4 (Sigma-Aldrich), 20 mM N-ethylmaleimide (NEM) (Thermo), 1 mM PMSF, 1.7% Triton X-100 (Roth), 1 mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich), 100 μM methyl arachidonyl fluorophosphonate (MAFP) (Sigma-Aldrich), cOmplete EDTA-free protease inhibitor cocktail (PI-cocktail) (Roche) at a protein concentration of 3 mg/ml, then spun at 16,100 × g, 4 °C, 10 min to remove cell debris. ..

Blue Native PAGE:

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects
Article Snippet: Paragraph title: Blue native PAGE and 2D gel analysis ... Mitochondria were solubilized in buffer (1% digitonin [Merck, 300410], 20 mM Tris-HCl, pH 7.4 [Roth, 9090.1], 0.1 mM EDTA, 50 mM NaCl [Roth, P029.5], 10% (w/v) glycerol [Roth, 7530.1], 1 mM PMSF, 10 mM NEM) to a final concentration of 1 µg/µL for 30 min at 4°C.

Mouse Assay:

Article Title: Protection of Batf3‐deficient mice from experimental cerebral malaria correlates with impaired cytotoxic T‐cell responses and immune regulation
Article Snippet: Preparation of organs and lymphocytes for ex vivo analysis Deeply anaesthetized mice (see above) were intracardially perfused with 1 × PBS for 5 min to remove circulating and non‐adhered blood leucocytes from the organs. .. The interphase containing the lymphocytes was carefully transferred into a new tube and washed with 1 × PBS/1% FCS (PAA, Cölbe, Germany)/2 mm EDTA (Roth, Karlsruhe, Germany).

SDS Page:

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects
Article Snippet: Mitochondria were solubilized in buffer (1% digitonin [Merck, 300410], 20 mM Tris-HCl, pH 7.4 [Roth, 9090.1], 0.1 mM EDTA, 50 mM NaCl [Roth, P029.5], 10% (w/v) glycerol [Roth, 7530.1], 1 mM PMSF, 10 mM NEM) to a final concentration of 1 µg/µL for 30 min at 4°C. .. For subsequent 2D analysis, the appropriate lanes were excised from the BN-PAGE gel and incubated in equilibration buffer (25 mM Tris, pH 8.5, 0.2 M glycine, 1% SDS [Roth, 5136.1] and 2% 1,4-dithiothreitol [Roth, 6908.1]) for 10 min prior to further separation by SDS-PAGE.

Plasmid Preparation:

Article Title: Viscoelastic properties of vimentin originate from nonequilibrium conformational changes
Article Snippet: One hundred microliters of thawed bacteria solution and 1 μl of plasmid solution (1 μg/μl; diluted 1:100) were mixed and incubated on ice for 5 min. Fifty microliters of this mixture was plated on an ampicillin-containing lysogeny broth agar plate (L5667, Sigma-Aldrich) and incubated at 37°C overnight. .. The pellet was transferred to a cooled douncer, homogenized with 16 ml of tris buffer (50 mM; pH 8.0; Carl Roth GmbH) containing saccharose (25%, w/v), EDTA (1 mM; Carl Roth GmbH), and lysozyme (10 mg/ml; Roche Diagnostics), and incubated on ice for 30 min.

Irradiation:

Article Title: Distinct Clones of Yersinia pestis Caused the Black Death
Article Snippet: After arrival in the laboratory, samples were submitted to decontamination procedures consisting of 45 minutes of UV irradiation on each side, mechanical removal of the outer surface by sandblasting (Harnisch und Rieth, Winterbach, Germany) and a second UV irradiation. .. Aliquots of 0.38–0.5 g powder were incubated overnight on a rotary mixer at 37°C in a decalcification and digestion solution consisting of 0.5 M EDTA (pH 8; Roth, Karlsruhe, Germany), 0.4% N-Laurylsarcosine and 0.46 mg/ml Proteinase K (Roche, Germany).

Sample Prep:

Article Title: Distinct Clones of Yersinia pestis Caused the Black Death
Article Snippet: Paragraph title: Sample preparation and DNA extraction ... Aliquots of 0.38–0.5 g powder were incubated overnight on a rotary mixer at 37°C in a decalcification and digestion solution consisting of 0.5 M EDTA (pH 8; Roth, Karlsruhe, Germany), 0.4% N-Laurylsarcosine and 0.46 mg/ml Proteinase K (Roche, Germany).

In Vitro:

Article Title: Protection of Batf3‐deficient mice from experimental cerebral malaria correlates with impaired cytotoxic T‐cell responses and immune regulation
Article Snippet: The interphase containing the lymphocytes was carefully transferred into a new tube and washed with 1 × PBS/1% FCS (PAA, Cölbe, Germany)/2 mm EDTA (Roth, Karlsruhe, Germany). .. Then, single cells were resuspended in RPMI medium (Sigma‐Aldrich, Munich, Germany) containing 10% fetal calf serum (PAA Laboratories GmbH, Cölbe, Germany), 1% penicillin/streptomycin (Lonza, Wuppertal, Germany), 1% l ‐glutamine (PAA), and used for either flow cytometric staining or in vitro incubation.

Homogenization:

Article Title: Viscoelastic properties of vimentin originate from nonequilibrium conformational changes
Article Snippet: The pellet was transferred to a cooled douncer, homogenized with 16 ml of tris buffer (50 mM; pH 8.0; Carl Roth GmbH) containing saccharose (25%, w/v), EDTA (1 mM; Carl Roth GmbH), and lysozyme (10 mg/ml; Roche Diagnostics), and incubated on ice for 30 min. .. Eight hundred microliters of MgCl2 solution (1 M; Sigma-Aldrich), 80 μl of deoxyribonuclease I (DNase 1; 50 mg/ml, Sigma-Aldrich) in tris buffer (10 mM; pH 7.5) containing NaCl (100 mM; Carl Roth GmbH), 80 μl of ribonuclease A (RNase A; 10 mg/ml; Roche Diagnostics) in tris buffer (100 mM; pH 7.5), 800 μl of saturated phenylmethylsulfonyl fluoride (PMSF; Serva) in ethanol, and 1.6 ml of 10% NP-40 (Roche Diagnostics) were added and mixed several times by homogenization.

Concentration Assay:

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects
Article Snippet: .. Mitochondria were solubilized in buffer (1% digitonin [Merck, 300410], 20 mM Tris-HCl, pH 7.4 [Roth, 9090.1], 0.1 mM EDTA, 50 mM NaCl [Roth, P029.5], 10% (w/v) glycerol [Roth, 7530.1], 1 mM PMSF, 10 mM NEM) to a final concentration of 1 µg/µL for 30 min at 4°C. .. Lysates were cleared by centrifugation at 14,000 g for 10 min at 4°C and 10X loading dye was added (5% Coomassie brilliant blue G-250 [Roth, 9598.2], 500 mM 6-aminohexanoic acid [Sigma-Aldrich, 07260], 100 mM Bis-Tris, pH 7.0).

Article Title: ?-Fucosidase as a novel convenient biomarker for cellular senescence
Article Snippet: .. To measure hydrolase activities, 5 µL of the supernatant were incubated in 45 µL of hydrolase-specific reaction buffer containing hydrolase-specific 4-MU-glycosides for 1 h at 37°C. α-L-fucosidase was measured in 0.2 M sodium citrate pH 4.5, using 1 mM 4-MU-α-L-fucoside (Sigma), β-galactosidase in 66 mM NaCl, 66 mM Na2 HPO4 , 33 mM sodium citrate, pH 4.6, using 1 mM 4-MU-β-D-galactoside (Roth) as described. β-glucuronidase was measured in 200 mM sodium acetate buffer, pH 5.0, 10 mM EDTA, 0.01% BSA, 0.1% Triton X-100 using 2.5 mM 4-MU-β-D-glucuronide (Roth) according to Sperker et al. Enzymatic reactions were stopped by the addition of 0.2 mL Na2 CO3 , and reaction mixtures were centrifuged for 10 min at 2,000 g. The cleared supernatant was used to determine the concentration of 4-MU by fluorescence detection (excitation 355 nm, emission 460 nm) using an Infinite 200 Reader (Tecan, Männedorf, Switzerland). ..

Cell Counting:

Article Title: A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells
Article Snippet: After removing the supernatant above the leukocyte layer, the latter was carefully collected with a plastic pipette and washed with washing buffer (PBS with 0.5% bovine serum albumin (Sigma-Aldrich, Steinheim, Germany) and 2 mM ethylenediaminetetraacetic acid (Carl Roth GmbH & Co. KG, Karlsruhe, Germany)) at 300 x g for 10 min (brakes: level two). .. The two leukocyte recovery protocols were compared by cell counting and flow cytometric analysis focusing on the forward scatter/ side scatter distribution of the cells.

Fractionation:

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects
Article Snippet: Paragraph title: Mitochondrial preparation and fractionation ... Cells were harvested in phosphate-buffered saline (PBS; 10 mM Na2 HPO4 [Roth, T876.1], 1.8 mM KH2 PO4 [Roth, 3904.2], 137 mM NaCl [Roth, P029.5], 2.7 mM KCl [Roth, P017.2]), and washed using isolation buffer (300 mM trehalose [Roth, 5151.3], 10 mM KCl [Roth, P017.2],; 10 mM HEPES [Roth, 9105.2], pH 7.4, 1 mM EDTA [Roth, 8040.1], 0.5 mM phenylmethylsulfonylfluoride [PMSF; Roth, 6367.1] and 10 mM N-ethylmaleimide [NEM; Sigma-Aldrich, 128287]).

Two-Dimensional Gel Electrophoresis:

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects
Article Snippet: Paragraph title: Blue native PAGE and 2D gel analysis ... Mitochondria were solubilized in buffer (1% digitonin [Merck, 300410], 20 mM Tris-HCl, pH 7.4 [Roth, 9090.1], 0.1 mM EDTA, 50 mM NaCl [Roth, P029.5], 10% (w/v) glycerol [Roth, 7530.1], 1 mM PMSF, 10 mM NEM) to a final concentration of 1 µg/µL for 30 min at 4°C.

Standard Deviation:

Article Title: The effect of endodontic treatment using different intracanal medicaments on periodontal attachment level in concurrent endodontic-periodontal lesions: A randomized controlled trial
Article Snippet: The sample size calculation determined that 16 patients per treatment arm would provide 80% power to detect a true difference of 1.0 mm between test and control using probing depth (PD) reduction in pockets > 5 mm as the primary outcome variable, assuming that the common standard deviation is 1.0 mm. .. Smear layer was removed by irrigating with 5.0 mL 17% EDTA (Roth International Ltd, Chicago, IL, USA) for 1 min followed by 5.0 mL 1% NaOCl, and the final irrigant being 5 ml of distilled water.

Staining:

Article Title: Protection of Batf3‐deficient mice from experimental cerebral malaria correlates with impaired cytotoxic T‐cell responses and immune regulation
Article Snippet: The interphase containing the lymphocytes was carefully transferred into a new tube and washed with 1 × PBS/1% FCS (PAA, Cölbe, Germany)/2 mm EDTA (Roth, Karlsruhe, Germany). .. Then, single cells were resuspended in RPMI medium (Sigma‐Aldrich, Munich, Germany) containing 10% fetal calf serum (PAA Laboratories GmbH, Cölbe, Germany), 1% penicillin/streptomycin (Lonza, Wuppertal, Germany), 1% l ‐glutamine (PAA), and used for either flow cytometric staining or in vitro incubation.

Gradient Centrifugation:

Article Title: A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells
Article Snippet: Aiming to preserve all subpopulations including granulocytes, at first, a protocol optimized for recovery of all leukocyte subpopulations was evaluated in comparison to standard density gradient centrifugation for peripheral blood mononuclear cells, using a Heraeus Multifuge X3R Centrifuge (Thermo Fisher Scientific, Darmstadt, Germany). .. After removing the supernatant above the leukocyte layer, the latter was carefully collected with a plastic pipette and washed with washing buffer (PBS with 0.5% bovine serum albumin (Sigma-Aldrich, Steinheim, Germany) and 2 mM ethylenediaminetetraacetic acid (Carl Roth GmbH & Co. KG, Karlsruhe, Germany)) at 300 x g for 10 min (brakes: level two).

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