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Effects of MLCK and myosin II inhibitors on restitution in gastroids. (A) Nuclei (Hoechst33342) or GFP (HuGE) images collected at the indicated times from a representative timecourse experiment using gastroids. Scale bar: <t>10â€Â</t> µm. A single
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1) Product Images from "Cell injury triggers actin polymerization to initiate epithelial restitution"

Article Title: Cell injury triggers actin polymerization to initiate epithelial restitution

Journal: Journal of Cell Science

doi: 10.1242/jcs.216317

Effects of MLCK and myosin II inhibitors on restitution in gastroids. (A) Nuclei (Hoechst33342) or GFP (HuGE) images collected at the indicated times from a representative timecourse experiment using gastroids. Scale bar: 10†µm. A single
Figure Legend Snippet: Effects of MLCK and myosin II inhibitors on restitution in gastroids. (A) Nuclei (Hoechst33342) or GFP (HuGE) images collected at the indicated times from a representative timecourse experiment using gastroids. Scale bar: 10†µm. A single

Techniques Used:

Effects of several inhibitors on restitution in gastroids. (A) Nuclei (Hoechst 33342) or GFP (HuGE) images collected at the indicated times from a representative timecourse experiment using gastroids. Scale bar: 10†µm. A single gastric
Figure Legend Snippet: Effects of several inhibitors on restitution in gastroids. (A) Nuclei (Hoechst 33342) or GFP (HuGE) images collected at the indicated times from a representative timecourse experiment using gastroids. Scale bar: 10†µm. A single gastric

Techniques Used:

Related Articles

Centrifugation:

Article Title: Multiple Layers of Phospho-Regulation Coordinate Metabolism and the Cell Cycle in Budding Yeast
Article Snippet: Cells were lysed by bead beating in 8M urea, 150 mM NaCl, 5 mM DTT, 50 mM HEPES pH 8 supplemented with 1x HaltTM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). .. The lysate was centrifuged at 13,200 rpm for 15 min and the supernatant was transferred to fresh test tubes for a second round of centrifugation.

Luciferase:

Article Title: The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection
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Construct:

Article Title: The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection
Article Snippet: All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Cellgro; Mediatech, Herndon, VA, USA) supplemented with 10% fetal calf serum (FCS) (Cellgro), 10 mM HEPES (Invitrogen, Carlsbad, CA, USA), 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine (Invitrogen) in 5% CO2 at 37 °C. .. The JFH-1 Rluc/SGR wt or JFH-1 Rluc/SGR GND plasmids carry bicistronic constructs containing the luciferase reporter gene in the first cistron and wild-type (wt) or replication-deficient (encoding a GDD-to-GND mutation in NS5B) JFH-1 sub-genomic replicon in the second cistron, respectively [ ].

Incubation:

Article Title: Cell injury triggers actin polymerization to initiate epithelial restitution
Article Snippet: Briefly, isolated mouse gastric corpus was incubated with rocking at 4°C for 2†h in 10†mM EDTA in Dulbecco's phosphate-buffered saline (DPBS) without Ca2+ and Mg2+ . .. After Matrigel polymerization at 37°C, advanced DMEM/F12 supplemented with 2†mM GlutaMax, 10†mM HEPES, 100†U/ml penicillin/100†μg/ml streptomycin, 1× N2 and 1× B27 supplements (Life Technologies), Wnt3a-conditioned medium (50%), R-spondin-conditioned medium (10%), Noggin-conditioned medium (10%), [Leu15]-Gastrin I (10†nM, Sigma) and EGF (50†ng/ml, PeproTech) was added to the wells and replaced every 4†days.

Article Title: Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges
Article Snippet: .. After dissection, the fat tissue was minced and incubated for 35 min at 37°C under gentle agitation (150 rpm) in complete Roswell Park Memorial Institute medium (cRPMI; 10% FBS, 1 mM of sodium pyruvate [Gibco; 11360-070], Glutamax 1× [Gibco; 35050-061], MEM nonessential amino acids [Gibco; 11140-050], Hepes [Gibco; 15630-080], penicillin/streptomycin [Gibco; 15140-122], and RPMI [Gibco; 21870-076] containing 1 mg/ml of collagenase type VIII [Sigma-Aldrich; C2139], and 100 µg/ml of DNase I [Sigma-Aldrich; 10104159001]). ..

Activity Assay:

Article Title: Multiple Layers of Phospho-Regulation Coordinate Metabolism and the Cell Cycle in Budding Yeast
Article Snippet: Sampling, Protein Extraction, and Digestion Twenty milliliter of cell culture (OD ∼0.6) were sampled into 1.5 volumes of 60% methanol and precooled to −40°C to quench metabolic activity. .. Cells were lysed by bead beating in 8M urea, 150 mM NaCl, 5 mM DTT, 50 mM HEPES pH 8 supplemented with 1x HaltTM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific).

Expressing:

Article Title: Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy
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Modification:

Article Title: The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection
Article Snippet: .. All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Cellgro; Mediatech, Herndon, VA, USA) supplemented with 10% fetal calf serum (FCS) (Cellgro), 10 mM HEPES (Invitrogen, Carlsbad, CA, USA), 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine (Invitrogen) in 5% CO2 at 37 °C. .. The sub-genomic and full-length JFH-1 stable replicon Huh-7 cell lines were cultured in medium supplemented with 400 or 200 µg/mL of G418, respectively, as described previously [ ].

Western Blot:

Article Title: Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection
Article Snippet: After one freeze-thaw cycle, VapBC-1 was isolated in accordance with the MagneHis protein purification system (Promega, Madison, WI) with the following exceptions: the lysis solution was 1× BugBuster protein extraction reagent (Millipore Sigma, Burlington, MA) with 100 mM HEPES (pH 7.2), 300 mM NaCl, 1× ProteCEASE-50 (EDTA free), and 2 μl/ml RNase-free DNase (Thermo Fisher Scientific, Waltham, MA); the wash buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 20 mM imidazole; and the elution buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 500 mM imidazole. .. One microgram of purified VapBC-1 was separated on a Bolt precast 4 to 12% polyacrylamide gel and analyzed via Western blotting using the iBlot and iBind system (Thermo Fisher Scientific, Waltham, MA).

Crystallization Assay:

Article Title: Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection
Article Snippet: Paragraph title: Purification of VapC-1 for crystallization studies. ... After one freeze-thaw cycle, VapBC-1 was isolated in accordance with the MagneHis protein purification system (Promega, Madison, WI) with the following exceptions: the lysis solution was 1× BugBuster protein extraction reagent (Millipore Sigma, Burlington, MA) with 100 mM HEPES (pH 7.2), 300 mM NaCl, 1× ProteCEASE-50 (EDTA free), and 2 μl/ml RNase-free DNase (Thermo Fisher Scientific, Waltham, MA); the wash buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 20 mM imidazole; and the elution buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 500 mM imidazole.

Transfection:

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Dissection:

Article Title: Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges
Article Snippet: .. After dissection, the fat tissue was minced and incubated for 35 min at 37°C under gentle agitation (150 rpm) in complete Roswell Park Memorial Institute medium (cRPMI; 10% FBS, 1 mM of sodium pyruvate [Gibco; 11360-070], Glutamax 1× [Gibco; 35050-061], MEM nonessential amino acids [Gibco; 11140-050], Hepes [Gibco; 15630-080], penicillin/streptomycin [Gibco; 15140-122], and RPMI [Gibco; 21870-076] containing 1 mg/ml of collagenase type VIII [Sigma-Aldrich; C2139], and 100 µg/ml of DNase I [Sigma-Aldrich; 10104159001]). ..

Cell Culture:

Article Title: The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection
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Article Title: Cell injury triggers actin polymerization to initiate epithelial restitution
Article Snippet: After Matrigel polymerization at 37°C, advanced DMEM/F12 supplemented with 2†mM GlutaMax, 10†mM HEPES, 100†U/ml penicillin/100†μg/ml streptomycin, 1× N2 and 1× B27 supplements (Life Technologies), Wnt3a-conditioned medium (50%), R-spondin-conditioned medium (10%), Noggin-conditioned medium (10%), [Leu15]-Gastrin I (10†nM, Sigma) and EGF (50†ng/ml, PeproTech) was added to the wells and replaced every 4†days. .. Gastroids were cultured in a 5% CO2 incubator at 37°C.

Article Title: Adenosine Depletion as A New Strategy to Decrease Glioblastoma Stem-Like Cells Aggressiveness
Article Snippet: .. GSCs primary culture were obtained from surgical human GBM samples and cultured with M21 medium DMEM/F12 (Gibco); 1X MEM non- essential amino acids (Gibco™); Hepes (1 M; Gibco); D-Glucose (45%; Sigma, Darmstadt, Germany); BSA Fraction V (7.5%; ThermoFisher); Sodium pyruvate (100 mM; ThermoFisher); L-glutamine (200 mM; Gibco); penicillin/streptomycin (100 U/mL, Gibco); hydrocortisone (0.6 µg/mL; Sigma); triiodothyronine (0.1 mg/mL; Sigma) and 1X N1 supplement (Sigma) supplemented with EGF (25 ng/μL; Sigma), bFGF (25 ng/μL; Sigma) and heparin (0.5 μg/mL; Sigma). normoxia (21% O2 ) and hypoxia (0.5% O2 ) conditions were generated using a gas chamber (5% CO2 and 95% N2 mixture) during 24 h. .. Adenosine Quantification GSCs were cultured and treated with ADA 1U/mL by 24 h. Culture medium was collected and deproteinization was performed with TCA and then filtered.

Article Title: Convection-enhanced delivery of temozolomide and whole cell tumor immunizations in GL261 and KR158 experimental mouse gliomas
Article Snippet: .. The cells were cultured at 37 °C in the presence of 5% CO2 in R10-medium containing: RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 50 μg/mL gentamicin (GIBCO-Life technologies) and 10% fetal bovine serum (Biochrom AG). ..

Article Title: Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy
Article Snippet: Paragraph title: Cell culture and cell transfection. ... THP-1 cells were acquired from the University of North Carolina (UNC) Lineberger Tissue Culture Facility (ATCC TIB-202, passage 25) and were maintained in RPMI 1640 medium containing 10% FBS, 1% penicillin/streptomycin antibiotics, 2 mM glutamine, 10 mM HEPES, and 1× nonessential amino acids (all from GIBCO).

Generated:

Article Title: Cell injury triggers actin polymerization to initiate epithelial restitution
Article Snippet: Gastroids from mouse gastric corpus were generated as described previously ( ; ). .. After Matrigel polymerization at 37°C, advanced DMEM/F12 supplemented with 2†mM GlutaMax, 10†mM HEPES, 100†U/ml penicillin/100†μg/ml streptomycin, 1× N2 and 1× B27 supplements (Life Technologies), Wnt3a-conditioned medium (50%), R-spondin-conditioned medium (10%), Noggin-conditioned medium (10%), [Leu15]-Gastrin I (10†nM, Sigma) and EGF (50†ng/ml, PeproTech) was added to the wells and replaced every 4†days.

Article Title: Adenosine Depletion as A New Strategy to Decrease Glioblastoma Stem-Like Cells Aggressiveness
Article Snippet: .. GSCs primary culture were obtained from surgical human GBM samples and cultured with M21 medium DMEM/F12 (Gibco); 1X MEM non- essential amino acids (Gibco™); Hepes (1 M; Gibco); D-Glucose (45%; Sigma, Darmstadt, Germany); BSA Fraction V (7.5%; ThermoFisher); Sodium pyruvate (100 mM; ThermoFisher); L-glutamine (200 mM; Gibco); penicillin/streptomycin (100 U/mL, Gibco); hydrocortisone (0.6 µg/mL; Sigma); triiodothyronine (0.1 mg/mL; Sigma) and 1X N1 supplement (Sigma) supplemented with EGF (25 ng/μL; Sigma), bFGF (25 ng/μL; Sigma) and heparin (0.5 μg/mL; Sigma). normoxia (21% O2 ) and hypoxia (0.5% O2 ) conditions were generated using a gas chamber (5% CO2 and 95% N2 mixture) during 24 h. .. Adenosine Quantification GSCs were cultured and treated with ADA 1U/mL by 24 h. Culture medium was collected and deproteinization was performed with TCA and then filtered.

other:

Article Title: Mechanisms of Calcium Decay Kinetics in Hippocampal Spines: Role of Spine Calcium Pumps and Calcium Diffusion through the Spine Neck in Biochemical Compartmentalization
Article Snippet: Electrodes were filled with a solution containing (in m m ): 5 NaCl, 10 KCl, 10 HEPES, 135 KMeSO4 , 2.5–4 Mg-ATP, 0.3 Na-GTP, and 200 μ m calcium green-1 (Molecular Probes, Eugene, OR); resistances were 6–7 MΩ .

Polymerase Chain Reaction:

Article Title: BCL6 corepressor contributes to Th17 cell formation by inhibiting Th17 fate suppressors
Article Snippet: The product was purified with a MinElute PCR Purification Kit (Qiagen), and a second golden gate reaction was performed using SapI (New England Biolabs) and T4 DNA ligase (New England Biolabs) to replace the CCDB gene in the pMCIA vector with the tRNA–gRNA arrays made from the first golden gate reaction. .. Platinum-E cells were grown in D10 media containing DMEM media (Gibco) supplemented with 10% FBS (Omega), penicillin/streptomycin (Life Technologies), Glutamax (Life Technologies), β-mercaptoethanol (Sigma), Hepes (Life Technologies), sodium pyruvate (Life Technologies), MEM nonessential amino acids (Life Technologies), and Plasmocin prophylactic (Invivogen).

Mutagenesis:

Article Title: The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection
Article Snippet: All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Cellgro; Mediatech, Herndon, VA, USA) supplemented with 10% fetal calf serum (FCS) (Cellgro), 10 mM HEPES (Invitrogen, Carlsbad, CA, USA), 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine (Invitrogen) in 5% CO2 at 37 °C. .. The JFH-1 Rluc/SGR wt or JFH-1 Rluc/SGR GND plasmids carry bicistronic constructs containing the luciferase reporter gene in the first cistron and wild-type (wt) or replication-deficient (encoding a GDD-to-GND mutation in NS5B) JFH-1 sub-genomic replicon in the second cistron, respectively [ ].

Isolation:

Article Title: Cell injury triggers actin polymerization to initiate epithelial restitution
Article Snippet: Briefly, isolated mouse gastric corpus was incubated with rocking at 4°C for 2†h in 10†mM EDTA in Dulbecco's phosphate-buffered saline (DPBS) without Ca2+ and Mg2+ . .. After Matrigel polymerization at 37°C, advanced DMEM/F12 supplemented with 2†mM GlutaMax, 10†mM HEPES, 100†U/ml penicillin/100†μg/ml streptomycin, 1× N2 and 1× B27 supplements (Life Technologies), Wnt3a-conditioned medium (50%), R-spondin-conditioned medium (10%), Noggin-conditioned medium (10%), [Leu15]-Gastrin I (10†nM, Sigma) and EGF (50†ng/ml, PeproTech) was added to the wells and replaced every 4†days.

Article Title: Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection
Article Snippet: .. After one freeze-thaw cycle, VapBC-1 was isolated in accordance with the MagneHis protein purification system (Promega, Madison, WI) with the following exceptions: the lysis solution was 1× BugBuster protein extraction reagent (Millipore Sigma, Burlington, MA) with 100 mM HEPES (pH 7.2), 300 mM NaCl, 1× ProteCEASE-50 (EDTA free), and 2 μl/ml RNase-free DNase (Thermo Fisher Scientific, Waltham, MA); the wash buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 20 mM imidazole; and the elution buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 500 mM imidazole. .. One microgram of purified VapBC-1 was separated on a Bolt precast 4 to 12% polyacrylamide gel and analyzed via Western blotting using the iBlot and iBind system (Thermo Fisher Scientific, Waltham, MA).

Mouse Assay:

Article Title: Convection-enhanced delivery of temozolomide and whole cell tumor immunizations in GL261 and KR158 experimental mouse gliomas
Article Snippet: Cell line and cell culture medium Glioma cell lines syngeneic with C57BL/6 mice were used in this study. .. The cells were cultured at 37 °C in the presence of 5% CO2 in R10-medium containing: RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 50 μg/mL gentamicin (GIBCO-Life technologies) and 10% fetal bovine serum (Biochrom AG).

Article Title: Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges
Article Snippet: Adipose tissue SVF purification To isolate leukocytes from adipose tissue, mice were anesthetized with a mixture containing 12.5 mg/ml ketamine, 2.5 mg/ml xylazine, and 25 mg/ml acepromazine and intracardially perfused with PBS with 5 mM EDTA. .. After dissection, the fat tissue was minced and incubated for 35 min at 37°C under gentle agitation (150 rpm) in complete Roswell Park Memorial Institute medium (cRPMI; 10% FBS, 1 mM of sodium pyruvate [Gibco; 11360-070], Glutamax 1× [Gibco; 35050-061], MEM nonessential amino acids [Gibco; 11140-050], Hepes [Gibco; 15630-080], penicillin/streptomycin [Gibco; 15140-122], and RPMI [Gibco; 21870-076] containing 1 mg/ml of collagenase type VIII [Sigma-Aldrich; C2139], and 100 µg/ml of DNase I [Sigma-Aldrich; 10104159001]).

Protein Purification:

Article Title: Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection
Article Snippet: .. After one freeze-thaw cycle, VapBC-1 was isolated in accordance with the MagneHis protein purification system (Promega, Madison, WI) with the following exceptions: the lysis solution was 1× BugBuster protein extraction reagent (Millipore Sigma, Burlington, MA) with 100 mM HEPES (pH 7.2), 300 mM NaCl, 1× ProteCEASE-50 (EDTA free), and 2 μl/ml RNase-free DNase (Thermo Fisher Scientific, Waltham, MA); the wash buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 20 mM imidazole; and the elution buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 500 mM imidazole. .. One microgram of purified VapBC-1 was separated on a Bolt precast 4 to 12% polyacrylamide gel and analyzed via Western blotting using the iBlot and iBind system (Thermo Fisher Scientific, Waltham, MA).

Protein Extraction:

Article Title: Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection
Article Snippet: .. After one freeze-thaw cycle, VapBC-1 was isolated in accordance with the MagneHis protein purification system (Promega, Madison, WI) with the following exceptions: the lysis solution was 1× BugBuster protein extraction reagent (Millipore Sigma, Burlington, MA) with 100 mM HEPES (pH 7.2), 300 mM NaCl, 1× ProteCEASE-50 (EDTA free), and 2 μl/ml RNase-free DNase (Thermo Fisher Scientific, Waltham, MA); the wash buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 20 mM imidazole; and the elution buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 500 mM imidazole. .. One microgram of purified VapBC-1 was separated on a Bolt precast 4 to 12% polyacrylamide gel and analyzed via Western blotting using the iBlot and iBind system (Thermo Fisher Scientific, Waltham, MA).

Article Title: Multiple Layers of Phospho-Regulation Coordinate Metabolism and the Cell Cycle in Budding Yeast
Article Snippet: Paragraph title: Sampling, Protein Extraction, and Digestion ... Cells were lysed by bead beating in 8M urea, 150 mM NaCl, 5 mM DTT, 50 mM HEPES pH 8 supplemented with 1x HaltTM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific).

CRISPR:

Article Title: BCL6 corepressor contributes to Th17 cell formation by inhibiting Th17 fate suppressors
Article Snippet: Paragraph title: CRISPR/Cas9 and retroviral transductions ... Platinum-E cells were grown in D10 media containing DMEM media (Gibco) supplemented with 10% FBS (Omega), penicillin/streptomycin (Life Technologies), Glutamax (Life Technologies), β-mercaptoethanol (Sigma), Hepes (Life Technologies), sodium pyruvate (Life Technologies), MEM nonessential amino acids (Life Technologies), and Plasmocin prophylactic (Invivogen).

Purification:

Article Title: BCL6 corepressor contributes to Th17 cell formation by inhibiting Th17 fate suppressors
Article Snippet: The product was purified with a MinElute PCR Purification Kit (Qiagen), and a second golden gate reaction was performed using SapI (New England Biolabs) and T4 DNA ligase (New England Biolabs) to replace the CCDB gene in the pMCIA vector with the tRNA–gRNA arrays made from the first golden gate reaction. .. Platinum-E cells were grown in D10 media containing DMEM media (Gibco) supplemented with 10% FBS (Omega), penicillin/streptomycin (Life Technologies), Glutamax (Life Technologies), β-mercaptoethanol (Sigma), Hepes (Life Technologies), sodium pyruvate (Life Technologies), MEM nonessential amino acids (Life Technologies), and Plasmocin prophylactic (Invivogen).

Article Title: Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection
Article Snippet: Paragraph title: Purification of VapC-1 for crystallization studies. ... After one freeze-thaw cycle, VapBC-1 was isolated in accordance with the MagneHis protein purification system (Promega, Madison, WI) with the following exceptions: the lysis solution was 1× BugBuster protein extraction reagent (Millipore Sigma, Burlington, MA) with 100 mM HEPES (pH 7.2), 300 mM NaCl, 1× ProteCEASE-50 (EDTA free), and 2 μl/ml RNase-free DNase (Thermo Fisher Scientific, Waltham, MA); the wash buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 20 mM imidazole; and the elution buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 500 mM imidazole.

Article Title: Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges
Article Snippet: Paragraph title: Adipose tissue SVF purification ... After dissection, the fat tissue was minced and incubated for 35 min at 37°C under gentle agitation (150 rpm) in complete Roswell Park Memorial Institute medium (cRPMI; 10% FBS, 1 mM of sodium pyruvate [Gibco; 11360-070], Glutamax 1× [Gibco; 35050-061], MEM nonessential amino acids [Gibco; 11140-050], Hepes [Gibco; 15630-080], penicillin/streptomycin [Gibco; 15140-122], and RPMI [Gibco; 21870-076] containing 1 mg/ml of collagenase type VIII [Sigma-Aldrich; C2139], and 100 µg/ml of DNase I [Sigma-Aldrich; 10104159001]).

Plasmid Preparation:

Article Title: The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection
Article Snippet: All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Cellgro; Mediatech, Herndon, VA, USA) supplemented with 10% fetal calf serum (FCS) (Cellgro), 10 mM HEPES (Invitrogen, Carlsbad, CA, USA), 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine (Invitrogen) in 5% CO2 at 37 °C. .. The JFH-1 genome-containing plasmid has been previously described [ ].

Article Title: BCL6 corepressor contributes to Th17 cell formation by inhibiting Th17 fate suppressors
Article Snippet: The product was purified with a MinElute PCR Purification Kit (Qiagen), and a second golden gate reaction was performed using SapI (New England Biolabs) and T4 DNA ligase (New England Biolabs) to replace the CCDB gene in the pMCIA vector with the tRNA–gRNA arrays made from the first golden gate reaction. .. Platinum-E cells were grown in D10 media containing DMEM media (Gibco) supplemented with 10% FBS (Omega), penicillin/streptomycin (Life Technologies), Glutamax (Life Technologies), β-mercaptoethanol (Sigma), Hepes (Life Technologies), sodium pyruvate (Life Technologies), MEM nonessential amino acids (Life Technologies), and Plasmocin prophylactic (Invivogen).

Sampling:

Article Title: Multiple Layers of Phospho-Regulation Coordinate Metabolism and the Cell Cycle in Budding Yeast
Article Snippet: Paragraph title: Sampling, Protein Extraction, and Digestion ... Cells were lysed by bead beating in 8M urea, 150 mM NaCl, 5 mM DTT, 50 mM HEPES pH 8 supplemented with 1x HaltTM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific).

Lysis:

Article Title: Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection
Article Snippet: .. After one freeze-thaw cycle, VapBC-1 was isolated in accordance with the MagneHis protein purification system (Promega, Madison, WI) with the following exceptions: the lysis solution was 1× BugBuster protein extraction reagent (Millipore Sigma, Burlington, MA) with 100 mM HEPES (pH 7.2), 300 mM NaCl, 1× ProteCEASE-50 (EDTA free), and 2 μl/ml RNase-free DNase (Thermo Fisher Scientific, Waltham, MA); the wash buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 20 mM imidazole; and the elution buffer contained 100 mM HEPES (pH 7.2), 300 mM NaCl, and 500 mM imidazole. .. One microgram of purified VapBC-1 was separated on a Bolt precast 4 to 12% polyacrylamide gel and analyzed via Western blotting using the iBlot and iBind system (Thermo Fisher Scientific, Waltham, MA).

Staining:

Article Title: Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges
Article Snippet: After dissection, the fat tissue was minced and incubated for 35 min at 37°C under gentle agitation (150 rpm) in complete Roswell Park Memorial Institute medium (cRPMI; 10% FBS, 1 mM of sodium pyruvate [Gibco; 11360-070], Glutamax 1× [Gibco; 35050-061], MEM nonessential amino acids [Gibco; 11140-050], Hepes [Gibco; 15630-080], penicillin/streptomycin [Gibco; 15140-122], and RPMI [Gibco; 21870-076] containing 1 mg/ml of collagenase type VIII [Sigma-Aldrich; C2139], and 100 µg/ml of DNase I [Sigma-Aldrich; 10104159001]). .. Although the RNAseq data shown in this article were obtained following the above extraction method, we also performed a comparative experiment analyzing VAM2, in which we used 2% FFA-free BSA (Thermo Fisher Scientific; BP9704100) in the digestion and staining buffers instead of FBS.

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    Thermo Fisher rpmi 1640 medium
    Conversion of Fe(III) to Fe(II) by Asc and thiols. All experiments were performed at 37 °C and used <t>RPMI-1640</t> medium supplemented with 50 mM HEPES, pH 7.4. (A) Time dependence of ferrozine-Fe(II) complex formation. Reactions contained 100 μM ferrozine and indicated concentrations of Fe(II) ammonium sulfate prepared as 1 mM stock in 10 mM ascorbic acid. (B) Fe(II) specificity of A562 absorbance by ferrozine (10 min incubations). Fe(II)–Fe(II) ammonium sulfate dissolved in ascorbic acid, Fe(III)–Fe(III) ammonium citrate dissolved in water. Data are means ± SD of triplicate measurements. (C) Time-course of Fe(II) formation from Fe(III) in the presence of 0.1 mM concentrations of reducers (ferrozine assay, means of triplicate measurements). The source of Fe(III) was Fe(III) ammonium citrate or Fe(III) chloride hexahydrate (both at 10 μM final concentrations, 1 mM stock solutions in H 2 O for Fe-citrate and in 10 mM HCl for FeCl 3 ). (D) Uptake of Cr(VI) by H460 cells in the presence of different extracellular reducers with or without 5 μM Fe(III) citrate (1 h uptake). Cell culture media contained 100 μM GSH, 50 μM Asc, or both reducers. Data are means ± SD, n = 3. Statistics: ***, p
    Rpmi 1640 Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher hepes buffer
    In vitro activity of <t>mitoDPP-3.</t> a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in <t>HEPES</t> (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1
    Hepes Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher hepes buffered saline hbs
    The OX42-immunogene forms large aggregates and triggers an immune response in microglia. (A) Comparison of the size-distribution profiles between complete cell culture medium (DMEM + 10% FBS) and the OX42-immunoporter obtained by DLS shows that the non-viral vehicle did not form aggregates. After adding pDNA to form the OX42-immunogene (N/P = 4), aggregates formed over a large range (≈50–1300 nm). (B) Adding FBS to polyplexes after maturation in <t>HBS</t> and maturation of polyplexes in FBS-containing medium caused a shift in size-distribution to lower aggregate sizes, but it had no effect on the width of size-distributions. Values are plotted as Mean ± SEM (two experiments). FBS, fetal bovine serum; OX42-IP, OX42-immunoporter; HBS, <t>HEPES-buffered</t> saline; D (+/–), DMEM with/without 10% FBS. (C) ROS production in microglial cells was measured by quantifying ROS-indicator fluorescence after 60 min and reported as percentage (%) of the internal standard PDBu. The aggregated OX42-immunogene triggered the respiratory burst (38.8 ± 3.3%) which was inhibited by the respiratory burst inhibitor diphenyliodonium (–7.02 ± 4.49%). The positive control zymosan caused the release of ROS at similar levels (45.8 ± 4.7%) to the OX42-immunogene. However, PEI–PEG (–0.97 ± 3.74%), OX42 antibody (2.57 ± 1.68%) and the OX42-immunoporter (9.83 ± 2.84%) did not trigger significant ROS production. Values are plotted as Mean ± SEM. ** P
    Hepes Buffered Saline Hbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Conversion of Fe(III) to Fe(II) by Asc and thiols. All experiments were performed at 37 °C and used RPMI-1640 medium supplemented with 50 mM HEPES, pH 7.4. (A) Time dependence of ferrozine-Fe(II) complex formation. Reactions contained 100 μM ferrozine and indicated concentrations of Fe(II) ammonium sulfate prepared as 1 mM stock in 10 mM ascorbic acid. (B) Fe(II) specificity of A562 absorbance by ferrozine (10 min incubations). Fe(II)–Fe(II) ammonium sulfate dissolved in ascorbic acid, Fe(III)–Fe(III) ammonium citrate dissolved in water. Data are means ± SD of triplicate measurements. (C) Time-course of Fe(II) formation from Fe(III) in the presence of 0.1 mM concentrations of reducers (ferrozine assay, means of triplicate measurements). The source of Fe(III) was Fe(III) ammonium citrate or Fe(III) chloride hexahydrate (both at 10 μM final concentrations, 1 mM stock solutions in H 2 O for Fe-citrate and in 10 mM HCl for FeCl 3 ). (D) Uptake of Cr(VI) by H460 cells in the presence of different extracellular reducers with or without 5 μM Fe(III) citrate (1 h uptake). Cell culture media contained 100 μM GSH, 50 μM Asc, or both reducers. Data are means ± SD, n = 3. Statistics: ***, p

    Journal: Chemical Research in Toxicology

    Article Title: Toxicological Antagonism among Welding Fume Metals: Inactivation of Soluble Cr(VI) by Iron

    doi: 10.1021/acs.chemrestox.8b00182

    Figure Lengend Snippet: Conversion of Fe(III) to Fe(II) by Asc and thiols. All experiments were performed at 37 °C and used RPMI-1640 medium supplemented with 50 mM HEPES, pH 7.4. (A) Time dependence of ferrozine-Fe(II) complex formation. Reactions contained 100 μM ferrozine and indicated concentrations of Fe(II) ammonium sulfate prepared as 1 mM stock in 10 mM ascorbic acid. (B) Fe(II) specificity of A562 absorbance by ferrozine (10 min incubations). Fe(II)–Fe(II) ammonium sulfate dissolved in ascorbic acid, Fe(III)–Fe(III) ammonium citrate dissolved in water. Data are means ± SD of triplicate measurements. (C) Time-course of Fe(II) formation from Fe(III) in the presence of 0.1 mM concentrations of reducers (ferrozine assay, means of triplicate measurements). The source of Fe(III) was Fe(III) ammonium citrate or Fe(III) chloride hexahydrate (both at 10 μM final concentrations, 1 mM stock solutions in H 2 O for Fe-citrate and in 10 mM HCl for FeCl 3 ). (D) Uptake of Cr(VI) by H460 cells in the presence of different extracellular reducers with or without 5 μM Fe(III) citrate (1 h uptake). Cell culture media contained 100 μM GSH, 50 μM Asc, or both reducers. Data are means ± SD, n = 3. Statistics: ***, p

    Article Snippet: H460 cells were grown in RPMI-1640 media (22400089, ThermoFisher) containing 10% (v/v) fetal bovine serum (FBS), and penicillin/streptomycin.

    Techniques: Ferrozine Assay, Cell Culture

    Effects of Fe(III) ions on Cr(VI) uptake in primary human bronchial epithelial cells. Cells were incubated for 1 h with 5 μM Cr(VI) for 1 h in RPMI-1640 medium supplemented with 50 mM HEPES (pH 7.4), growth factors, 50 μM Asc, 100 μM GSH and 0–10 μM Fe(III) citrate. (A) Suppression of Cr(VI) uptake by extracellular Fe(III). Data are means ± SD,; n = 3; ****, p

    Journal: Chemical Research in Toxicology

    Article Title: Toxicological Antagonism among Welding Fume Metals: Inactivation of Soluble Cr(VI) by Iron

    doi: 10.1021/acs.chemrestox.8b00182

    Figure Lengend Snippet: Effects of Fe(III) ions on Cr(VI) uptake in primary human bronchial epithelial cells. Cells were incubated for 1 h with 5 μM Cr(VI) for 1 h in RPMI-1640 medium supplemented with 50 mM HEPES (pH 7.4), growth factors, 50 μM Asc, 100 μM GSH and 0–10 μM Fe(III) citrate. (A) Suppression of Cr(VI) uptake by extracellular Fe(III). Data are means ± SD,; n = 3; ****, p

    Article Snippet: H460 cells were grown in RPMI-1640 media (22400089, ThermoFisher) containing 10% (v/v) fetal bovine serum (FBS), and penicillin/streptomycin.

    Techniques: Incubation

    Metabolism and toxicity of Cr(VI) in the presence of Ni(II) and Mn(II) ions. H460 cells were treated with Cr(VI) in the complete cell culture medium (RPMI-1640, 50 mM HEPES, pH 7.4, 10% FBS) additionally containing 50 μM Asc and 100 μM GSH. Reduction kinetics of Cr(VI) was measured at 37 °C. (A) Cellular uptake of Cr(VI) in the presence of Ni(II) or Mn(II). Cells were incubated with 10 μM Cr(VI) for 1 h. Data are means ± SD ( n = 3). (B) Accumulation of Cr by cells preincubated with Ni/Mn ions for 2 h prior to the addition of Cr(VI) (1 h uptake). Data are means ± SD ( n = 3). (C) Kinetics of Cr(VI) reduction by Asc/GSH in HEPES-supplemented RPMI-1640 medium. Samples contained 100 μM Asc, 200 μM GSH, 20 μM Cr(VI) and 0, 5, or 20 μM Ni(II) or Mn(II). (D) Viability of cells treated with Cr(VI) in the presence of 20 μM Ni(II) or Mn(II) ions. Cells were treated with metals for 3 h followed by 48 h recovery prior to cytotoxicity measurements. Data are means ± SD ( n = 3). (E) Kinetics of Cr(VI) reduction in HEPES buffer (100 mM HEPES, pH 7.4, 50 mM NaCl) in the presence of 0.2 mM Asc or (F) 2 mM GSH. Reactions contained 20 μM Cr(VI). Graphs show means of triplicate measurements.

    Journal: Chemical Research in Toxicology

    Article Title: Toxicological Antagonism among Welding Fume Metals: Inactivation of Soluble Cr(VI) by Iron

    doi: 10.1021/acs.chemrestox.8b00182

    Figure Lengend Snippet: Metabolism and toxicity of Cr(VI) in the presence of Ni(II) and Mn(II) ions. H460 cells were treated with Cr(VI) in the complete cell culture medium (RPMI-1640, 50 mM HEPES, pH 7.4, 10% FBS) additionally containing 50 μM Asc and 100 μM GSH. Reduction kinetics of Cr(VI) was measured at 37 °C. (A) Cellular uptake of Cr(VI) in the presence of Ni(II) or Mn(II). Cells were incubated with 10 μM Cr(VI) for 1 h. Data are means ± SD ( n = 3). (B) Accumulation of Cr by cells preincubated with Ni/Mn ions for 2 h prior to the addition of Cr(VI) (1 h uptake). Data are means ± SD ( n = 3). (C) Kinetics of Cr(VI) reduction by Asc/GSH in HEPES-supplemented RPMI-1640 medium. Samples contained 100 μM Asc, 200 μM GSH, 20 μM Cr(VI) and 0, 5, or 20 μM Ni(II) or Mn(II). (D) Viability of cells treated with Cr(VI) in the presence of 20 μM Ni(II) or Mn(II) ions. Cells were treated with metals for 3 h followed by 48 h recovery prior to cytotoxicity measurements. Data are means ± SD ( n = 3). (E) Kinetics of Cr(VI) reduction in HEPES buffer (100 mM HEPES, pH 7.4, 50 mM NaCl) in the presence of 0.2 mM Asc or (F) 2 mM GSH. Reactions contained 20 μM Cr(VI). Graphs show means of triplicate measurements.

    Article Snippet: H460 cells were grown in RPMI-1640 media (22400089, ThermoFisher) containing 10% (v/v) fetal bovine serum (FBS), and penicillin/streptomycin.

    Techniques: Cell Culture, Incubation

    Cr(VI) uptake and metal toxicity in HBEC3-KT cells. (A) Accumulation of Cr in cells after 1 h incubation in RPMI-1640 medium containing 50 mM HEPES (pH 7.4), 10% FBS, 100 μM GSH, 50 μM Asc, 5 μM Cr(VI) and 0–10 μM Fe(III) citrate. Data are means ± SD; n = 3; **, p

    Journal: Chemical Research in Toxicology

    Article Title: Toxicological Antagonism among Welding Fume Metals: Inactivation of Soluble Cr(VI) by Iron

    doi: 10.1021/acs.chemrestox.8b00182

    Figure Lengend Snippet: Cr(VI) uptake and metal toxicity in HBEC3-KT cells. (A) Accumulation of Cr in cells after 1 h incubation in RPMI-1640 medium containing 50 mM HEPES (pH 7.4), 10% FBS, 100 μM GSH, 50 μM Asc, 5 μM Cr(VI) and 0–10 μM Fe(III) citrate. Data are means ± SD; n = 3; **, p

    Article Snippet: H460 cells were grown in RPMI-1640 media (22400089, ThermoFisher) containing 10% (v/v) fetal bovine serum (FBS), and penicillin/streptomycin.

    Techniques: Incubation

    Effects of Fe ions on Cr(VI) metabolism. (A) Rates of Cr(VI) reduction at 37 °C in the presence of Fe(III). Reactions contained RPMI-1640 medium, 50 mM HEPES (pH 7.4), 20 μM Cr(VI), 0–20 μM Fe(III) citrate and a mixture of reducers (100 μM Asc, 200 μM GSH, 40 μM cysteine). Data are means of triplicates measurements. SD values were

    Journal: Chemical Research in Toxicology

    Article Title: Toxicological Antagonism among Welding Fume Metals: Inactivation of Soluble Cr(VI) by Iron

    doi: 10.1021/acs.chemrestox.8b00182

    Figure Lengend Snippet: Effects of Fe ions on Cr(VI) metabolism. (A) Rates of Cr(VI) reduction at 37 °C in the presence of Fe(III). Reactions contained RPMI-1640 medium, 50 mM HEPES (pH 7.4), 20 μM Cr(VI), 0–20 μM Fe(III) citrate and a mixture of reducers (100 μM Asc, 200 μM GSH, 40 μM cysteine). Data are means of triplicates measurements. SD values were

    Article Snippet: H460 cells were grown in RPMI-1640 media (22400089, ThermoFisher) containing 10% (v/v) fetal bovine serum (FBS), and penicillin/streptomycin.

    Techniques:

    Effects of Fe(II/III) chelators on Cr(VI) reduction in iron-rich and iron-poor cell culture media. Kinetics of Cr(VI) reduction in F-12K (iron-rich) and RPMI-1640 (iron-poor) media were measured as described in Figure 2 . Reactions contained 50 μM Cr(VI), indicated reducer and solvent control or one of Fe chelators (200 μM deferoxamine (DFX), 200 μM o -phenanthroline (o-PT) or 5 mM DTPA). (A) Reduction of Cr(VI) in the presence of 2 mM Cys or (B) 0.5 mM Asc. Plots are based on means of triplicate measurements taken every 20 s.

    Journal: Chemical Research in Toxicology

    Article Title: Toxicological Antagonism among Welding Fume Metals: Inactivation of Soluble Cr(VI) by Iron

    doi: 10.1021/acs.chemrestox.8b00182

    Figure Lengend Snippet: Effects of Fe(II/III) chelators on Cr(VI) reduction in iron-rich and iron-poor cell culture media. Kinetics of Cr(VI) reduction in F-12K (iron-rich) and RPMI-1640 (iron-poor) media were measured as described in Figure 2 . Reactions contained 50 μM Cr(VI), indicated reducer and solvent control or one of Fe chelators (200 μM deferoxamine (DFX), 200 μM o -phenanthroline (o-PT) or 5 mM DTPA). (A) Reduction of Cr(VI) in the presence of 2 mM Cys or (B) 0.5 mM Asc. Plots are based on means of triplicate measurements taken every 20 s.

    Article Snippet: H460 cells were grown in RPMI-1640 media (22400089, ThermoFisher) containing 10% (v/v) fetal bovine serum (FBS), and penicillin/streptomycin.

    Techniques: Cell Culture

    In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1

    Journal: Nature Communications

    Article Title: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

    doi: 10.1038/s41467-017-02655-1

    Figure Lengend Snippet: In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1

    Article Snippet: In total 150 µL of 2 µM mitoDPP-2 in HEPES buffer (20 mM, 150 mM NaCl, pH = 8.0) were added to a 96-well optical bottom plate (Nunc 265301, Thermo Scientific) at room temperature.

    Techniques: In Vitro, Activity Assay, Fluorescence, Purification

    Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1

    Journal: Nature Communications

    Article Title: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

    doi: 10.1038/s41467-017-02655-1

    Figure Lengend Snippet: Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1

    Article Snippet: In total 150 µL of 2 µM mitoDPP-2 in HEPES buffer (20 mM, 150 mM NaCl, pH = 8.0) were added to a 96-well optical bottom plate (Nunc 265301, Thermo Scientific) at room temperature.

    Techniques: In Vitro, Activity Assay, Fluorescence, Purification

    In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1

    Journal: Nature Communications

    Article Title: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

    doi: 10.1038/s41467-017-02655-1

    Figure Lengend Snippet: In vitro activity of mitoDPP-3. a Structure of mitoDPP-3. b In vitro fluorescence assay of 1 µM mitoDPP-3 in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). For plots, n = 3, error bars are ± s.e.m. c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-3 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-3 shows UV–vis absorbance at 300 nm with extinction coefficient 12.6 × 10 3 M −1 cm −1

    Article Snippet: In vitro kinetic assays of mitoDPP-2 and mitoDPP-3 In total 100 µL of 3 µM mitoDPP-2 or mitoDPP-3 in HEPES buffer (20 mM, pH = 7.4, 150 mM NaCl, 0.1% Triton X-100) were added to a 96-well optical bottom plate (Nunc 265301, Thermo Scientific) at room temperature.

    Techniques: In Vitro, Activity Assay, Fluorescence, Purification

    Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1

    Journal: Nature Communications

    Article Title: Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

    doi: 10.1038/s41467-017-02655-1

    Figure Lengend Snippet: Synthesis and in vitro activity of mitoDPP-2. a Synthetic scheme for mitoDPP-2. b In vitro fluorescence assay of mitoDPP-2 (1 µM) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with either 50 nM purified APT1 or APT2 (λ ex 490/20 nm, λ em 545/20 nm). Error bars are ± s.e.m. ( n = 3). c Fluorescence emission spectra at 30 min from probes as treated in ( b ). d UV–vis spectra of 25 µM mitoDPP-2 (black; normalized at 275 nm) and deprotected fluorophore product (gray; normalized at 513 nm) in HEPES (20 mM, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). MitoDPP-2 shows UV–vis absorbance at 300 nm with extinction coefficient 8.7 × 10 3 M −1 cm −1 . The deprotected fluorophore product shows a major UV-Vis absorbance peak at 513 nm with extinction coefficient 11.8 × 10 3 M −1 cm −1

    Article Snippet: In vitro kinetic assays of mitoDPP-2 and mitoDPP-3 In total 100 µL of 3 µM mitoDPP-2 or mitoDPP-3 in HEPES buffer (20 mM, pH = 7.4, 150 mM NaCl, 0.1% Triton X-100) were added to a 96-well optical bottom plate (Nunc 265301, Thermo Scientific) at room temperature.

    Techniques: In Vitro, Activity Assay, Fluorescence, Purification

    The OX42-immunogene forms large aggregates and triggers an immune response in microglia. (A) Comparison of the size-distribution profiles between complete cell culture medium (DMEM + 10% FBS) and the OX42-immunoporter obtained by DLS shows that the non-viral vehicle did not form aggregates. After adding pDNA to form the OX42-immunogene (N/P = 4), aggregates formed over a large range (≈50–1300 nm). (B) Adding FBS to polyplexes after maturation in HBS and maturation of polyplexes in FBS-containing medium caused a shift in size-distribution to lower aggregate sizes, but it had no effect on the width of size-distributions. Values are plotted as Mean ± SEM (two experiments). FBS, fetal bovine serum; OX42-IP, OX42-immunoporter; HBS, HEPES-buffered saline; D (+/–), DMEM with/without 10% FBS. (C) ROS production in microglial cells was measured by quantifying ROS-indicator fluorescence after 60 min and reported as percentage (%) of the internal standard PDBu. The aggregated OX42-immunogene triggered the respiratory burst (38.8 ± 3.3%) which was inhibited by the respiratory burst inhibitor diphenyliodonium (–7.02 ± 4.49%). The positive control zymosan caused the release of ROS at similar levels (45.8 ± 4.7%) to the OX42-immunogene. However, PEI–PEG (–0.97 ± 3.74%), OX42 antibody (2.57 ± 1.68%) and the OX42-immunoporter (9.83 ± 2.84%) did not trigger significant ROS production. Values are plotted as Mean ± SEM. ** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b

    doi: 10.3389/fnmol.2014.00079

    Figure Lengend Snippet: The OX42-immunogene forms large aggregates and triggers an immune response in microglia. (A) Comparison of the size-distribution profiles between complete cell culture medium (DMEM + 10% FBS) and the OX42-immunoporter obtained by DLS shows that the non-viral vehicle did not form aggregates. After adding pDNA to form the OX42-immunogene (N/P = 4), aggregates formed over a large range (≈50–1300 nm). (B) Adding FBS to polyplexes after maturation in HBS and maturation of polyplexes in FBS-containing medium caused a shift in size-distribution to lower aggregate sizes, but it had no effect on the width of size-distributions. Values are plotted as Mean ± SEM (two experiments). FBS, fetal bovine serum; OX42-IP, OX42-immunoporter; HBS, HEPES-buffered saline; D (+/–), DMEM with/without 10% FBS. (C) ROS production in microglial cells was measured by quantifying ROS-indicator fluorescence after 60 min and reported as percentage (%) of the internal standard PDBu. The aggregated OX42-immunogene triggered the respiratory burst (38.8 ± 3.3%) which was inhibited by the respiratory burst inhibitor diphenyliodonium (–7.02 ± 4.49%). The positive control zymosan caused the release of ROS at similar levels (45.8 ± 4.7%) to the OX42-immunogene. However, PEI–PEG (–0.97 ± 3.74%), OX42 antibody (2.57 ± 1.68%) and the OX42-immunoporter (9.83 ± 2.84%) did not trigger significant ROS production. Values are plotted as Mean ± SEM. ** P

    Article Snippet: OX42-IMMUNOPORTER CONJUGATION OX42 antibody in HEPES-buffered saline (HBS), pH 7.9, was modified with 10 mM N -Succinimidyl 3-(2-pyridyldithio)-propionate NHS ester (SPDP, ThermoFisher) at a ratio of 1.3 μL SPDP/mg OX42 for 2 h at room temperature.

    Techniques: Cell Culture, Fluorescence, Positive Control