Dissected tissue fragments were washed twice with 0.9% NaCl (0°C) and thereafter twice with 20 mM Krebs-HEPES buffer (Sigma-Aldrich Co., St Louis, MO, USA; 0°C, pH 7.4). Subsequently, the tissue fragments were placed in liquid nitrogen, homogenized, and dissolved in lysis buffer containing 5 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 0.2% carrier ampholyte. The samples prepared in that manner were then stored (−80°C) until further analysis.
Krebs hepes buffer
Manufactured by Merck Group
Sourced in United States
Krebs-Hepes buffer is a widely used cell culture medium component that helps maintain the physiological pH and osmolarity of cell culture systems. It is a buffer solution containing a combination of inorganic salts, amino acids, and other organic compounds that closely mimic the ionic composition of the human body. This buffer system helps to maintain a stable and optimal environment for the growth and maintenance of cells in vitro.
Automatically generated - may contain errors
3 protocols using krebs hepes buffer
1
Tissue Preparation for Proteomic Analysis
2
Quantifying Vascular O2⁻ and NO· Levels
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The method for O2– detection was previously described [24 (link)]. The femoral arteries isolated from all mice were dissected and allowed to equilibrate in deferoxamine-chelated Krebs-Hepes solution containing 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidin (CMH; 500 mM, Noxygen, Mainz, Germany), deferoxamine (25 mM, Sigma-Aldrich), and diethyldithiocarbamate (DETC; 5 mM, Sigma-Aldrich) at 37°C) for 45 min. The arteries were then frozen using liquid nitrogen.
NO· detection was performed using DETC as a spin trap as previously described [24 (link)]. The isolated femoral arteries were incubated for 45 min in a solution containing Krebs-Hepes buffer (BSA, 20.5 g/L, Sigma Aldrich), CaCl2 (3 mM), and L-arginine (0.8 mM, Sigma-Aldrich). Diethyldithiocarbamate-iron(II) complex (Fe[DETC]2) solution was added to the vessel and incubated for 45 min at 37°C. The arteries were then immediately frozen in plastic tubes using liquid nitrogen.
Both O2− and NO· measurements were performed on a table-top x-band spectrometer miniscope (MS200; Magnettech, Berlin, Germany). Recordings were made at 77°K, using a Dewar flask. Instrument settings were 10 mW of microwave power, 1 mT of amplitude modulation, 100 kHz of modulation frequency, 180 s of sweep time, and 4 scans. Values are expressed as the amplitude of signal per mg weight of dried femoral artery.
NO· detection was performed using DETC as a spin trap as previously described [24 (link)]. The isolated femoral arteries were incubated for 45 min in a solution containing Krebs-Hepes buffer (BSA, 20.5 g/L, Sigma Aldrich), CaCl2 (3 mM), and L-arginine (0.8 mM, Sigma-Aldrich). Diethyldithiocarbamate-iron(II) complex (Fe[DETC]2) solution was added to the vessel and incubated for 45 min at 37°C. The arteries were then immediately frozen in plastic tubes using liquid nitrogen.
Both O2− and NO· measurements were performed on a table-top x-band spectrometer miniscope (MS200; Magnettech, Berlin, Germany). Recordings were made at 77°K, using a Dewar flask. Instrument settings were 10 mW of microwave power, 1 mT of amplitude modulation, 100 kHz of modulation frequency, 180 s of sweep time, and 4 scans. Values are expressed as the amplitude of signal per mg weight of dried femoral artery.
3
Quantifying Nitric Oxide Production
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The detection of NO production was performed using the technique with Fe2+ diethyldithiocarbamate (DETC, Sigma Aldrich) as spin trap. Isolated heart and liver from mice injected with LPS, LPS with MVsnmMLCK+/+ or MVsnmMLCK-/-, and vehicle were incubated for 45 min in Krebs-Hepes buffer [bovine serum albumin (20.5 g/l), CaCl2 (3 mM) and L -arginine (0.8 mM); Sigma-Aldrich] and after treated with 250 μl of colloid Fe(DETC)2 and incubated at 37°C for 45 min (Ralay Ranaivo et al., 2007 (link); Leonetti et al., 2013 (link)). The organs were immediately frozen in plastic tubes. NO measurements were performed on a tabletop x-band spectrometer miniscope (MS200; Magnettech, Berlin, Germany). Values are expressed as amplitude of signal per weight of dried tissue.
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