Liposomes were prepared according to the method described by Kolasinac et al. with few modifications [25 (link)]. In brief, lipid components, like neutral and cationic lipids, and the fluorescent compound were mixed in Chloroform (EMSURE grade, VWR, Darmstadt, Germany) at a ratio of 1/1/0.1 mol/mol. Chloroform was evaporated under vacuum for 0.5 h. Afterward, lipids were dispersed in 20 mM N-2-hydroxyethylpiperazine-N-2 ethane sulfonic acid (HEPES) buffer (VWR, Darmstadt, Germany) at a total lipid concentration of 2 mg/mL and pH 7.4 (osmolality 30 mOsm/kg) or in distilled and deionized water at the same concentration. Buffer osmolality was determined using a freezing point osmometer (Osmomat 030 from Gonotec, Berlin, Germany). The solution was vortexed for 1–2 min to produce multilamellar liposomes. After homogenization in an ultrasonic bath (Sonocool, Bandelin electronic GmbH, Berlin, Germany) for 20 min at 5 °C, mainly unilamellar vesicles were formed. Before usage, liposomes were kept at 4 °C for no longer than two days.
Hepes buffer
Manufactured by Avantor
Sourced in Germany
HEPES buffer is a commonly used reagent in biological research and laboratory applications. It is a zwitterionic buffering agent that maintains a stable pH range, typically between 6.8 and 8.2, and is compatible with a variety of cell culture and biochemical assays. HEPES buffer helps to maintain a consistent and physiologically relevant environment for experiments.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
High glucose dmem, by Thermo Fisher Scientific (2 mentions)
Amphotericin b, by Thermo Fisher Scientific (2 mentions)
Chloroform, by Avantor (1 mentions)
Osmomat 030, by Gonotec (1 mentions)
Sonocool, by Bandelin (1 mentions)
Fetal bovine serum (fbs), by Capricorn (1 mentions)
Clindamycin phosphate, by Merck Group (1 mentions)
Diltiazem hydrochloride, by Merck Group (1 mentions)
Lidocaine, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Kemika (1 mentions)
Sodium chloride (nacl), by Kemika (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Catalase assay kit, by Cayman Chemical (1 mentions)
Superoxide dismutase assay kit, by Cayman Chemical (1 mentions)
7 protocols using hepes buffer
1
Preparation of Fluorescent Liposomes
2
Adherent Cell Staining with Fusogenic Liposomes
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To stain adherent cells, fusogenic liposomes (FLs) were used. Liposomes were produced as described in [47 (link)]. Briefly, DOPE, DOTAP, and fluorescently labeled lipid or membrane tracer molecules were mixed in chloroform at a weight ratio of DOPE/DOTAP/fluorescent dye of 1/1/0.05–0.1. Chloroform was evaporated in vacuum and the dried lipids were immediately dispersed in 20 mM HEPES buffer (pH 7.4; VWR, Darmstadt, Germany), at a total lipid concentration of 2 mg/mL. The solution was vortexed for 1–2 min and subsequently sonicated for 10 min. Before cell staining, FLs were diluted 1:100 in F10 Ham’s medium without supplements. Adherent cells were washed twice with warm PBS, the staining solution was applied at room temperature, and samples were placed at 37 °C. Fusion efficiencies between 50% and 90% of stained adherent cells were achieved regularly after 10 min incubation. The culture dish was washed again to remove unfused liposomes and refilled with warm myocyte growth medium.
3
Routine HeLa Cell Culture Conditions
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HeLa cells were routinely cultured at 37°C under an atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, high glucose, Gibco) supplemented with 10% fetal bovine serum (Capricorn Scientific, FBS-12A), 25 mM Hepes buffer (VWR), and antibiotics [100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), and 250 ng/ml Amphotericin B (Gibco)].
4
HeLa Cell Culture Conditions
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HeLa cells were routinely cultured at 37 °C under an atmosphere of 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, high glucose, Gibco) supplemented with 10% fetal bovine serum (Capricorn Scientific), 25 mM HEPES buffer (VWR), and antibiotics [100 U/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), and 250 ng/mL amphotericin B (Gibco)].
5
Binding Interactions of AAG with Drugs
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Native human AAG (#SLCD7253) and drug compounds (clindamycin phosphate, diltiazem hydrochloride and lidocaine) were purchased from Sigma–Aldrich (Darmstadt, Germany). Warfarin sodium salt was a gift from the Department of Medicinal Chemistry, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia. All chemicals were used without further purification. Immobilized SialEXO® (derived from Akkermansia muciniphilla and expressed in E. Coli) was obtained from Smart Enzymes™ Genovis (Lund, Sweden), and HEPES buffer was obtained from VWR International (Leuven, Belgium). All other reagents were of analytical grade or better. All solutions were prepared immediately before the experiments in order to minimize contamination and/or impair the stability of solutions. The solutions for microcalorimetric titrations were prepared in 25 mM HEPES at pH 7.40 containing 150 mM NaCl (Kemika, Zagreb, Croatia), using doubly distilled water and 5 M NaOH (Kemika, Zagreb, Croatia) for adjusting the pH value. AAG and drug solutions were prepared by dissolving the appropriate amount of solid in the given solvent.
6
Lymphocyte Proliferation Assay Protocol
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At 21 D and 42 D of age, 1 bird randomly from each replicate group was fasted for 12 h. Heparinized blood samples were collected from the wing vein. Lymphocytes were isolated from peripheral blood using lymphocyte density-gradient centrifugation medium (HaoYang Biological Manufacture Co. Ltd., Tianjin, China) and centrifuged at 1100 × g for 30 min. The lymphocyte fraction was collected from the interface and washed three times with RPMI-1640 culture medium (Invitrogen Corp., Grand Island, NY). After the final washing, cells were re-suspended in RPMI-1640 complete culture medium supplemented with 5% (vol/vol) heat inactivated fetal calf serum, 100 U penicillin/mL, 100 μg streptomycin/mL, and 24 mM Hepes buffer (Amresco 0511; Amresco Inc., Cleveland, OH). Cell viability was determined by the trypan blue exclusion method. Cell concentration was adjusted to 1 × 107 cells/mL culture medium. Subsequently, the number of proliferation peripheral blood T and B lymphocyte were calculated using the following equation: SI = OD570 (T/B lymphocyte proliferation group)/OD570 (control group).
7
Antioxidant Enzyme Assays in Violacein-Treated Cells
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Violacein-exposed cells were washed twice by resuspension in cold PBS, pH 7.4, and the cell pellets were sonicated in a cold buffer (20 mM HEPES buffer (Amresco, USA), pH 7.2, containing 1 mM EGTA (ethylene glycol tetraacetic acid; Sigma-Aldrich, USA), 210 mM mannitol and 70 mM sucrose) using the Ultrasonic processor (Sonic vibraCellTM, Canada). SOD activity was evaluated as indicated in the Superoxide Dismutase Assay Kit (Catalog No.706002; Cayman Chemical Company, USA). For catalase activity measurements, cells were treated, washed twice in PBS, pH 7.4, sonicated in a cold buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA) and tested for enzymatic activity as indicated in the Catalase Assay Kit (Catalog No.707002; Cayman Chemical Company). Protein quantification was determined by the Bradford assay [32 (link)].
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