According to the instructions of NP-40 lysis buffer (Beyotime, China), the corresponding volume of protease inhibitor PMSF (Beyotime, China) was added into NP-40 lysis buffer to ensure that the final concentration of PMSF was 1 mM. The frozen intestinal tissue was taken out from the refrigerator at -80°C. 100 μl NP-40 lysis buffer containing PMSF was added per 10 mg intestinal tissue and homogenized with ice electric homogenizer for 10 min; then, the tissue homogenate was centrifuged at 4°C (14000g, 10 min). The supernatant was taken and stored at -80°C. The concentrations of high-mobility group box-1 (HMGB1), interleukin- (IL-) 10, tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-8 in the supernatant of mouse intestinal tissue were measured with mouse HMGB1 (Arigobio, China) and IL-10, TNF-α, IL-1β, IL-6, and IL-8 ELISA kits (4A BIOTECH, China) and a BioTek Synergy H1 instrument (USA) following the manufacturers' directions.
Synergy h1 instrument
Manufactured by Agilent Technologies
Sourced in United States
The Synergy H1 instrument is a multi-mode microplate reader designed for a variety of applications in life science research. The instrument is capable of measuring absorbance, fluorescence, and luminescence signals in microplates.
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Lab products found in correlation
4 protocols using synergy h1 instrument
1
Quantification of Intestinal Inflammatory Mediators
2
Measuring Astrocyte Phagocytic Activity
3
Protein Stability Determination by Tryptophan Fluorescence
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A tryptophan fluorescence-based protein denaturation assay was performed to determine the protein stability of YiiM. The BL21-expressed YiiM protein was incubated in 20 mM Tris, pH 8.0, and 150 mM NaCl in the presence of urea (ecYiiM, 0.97–7.72 M; gsYiiM, 0.24–5.79 M) at room temperature for 30 min. The intensity of tryptophan fluorescence emitted at 325 nm was measured using a Synergy H1 instrument (BioTek) with an excitation wavelength of 295 nm.
4
Measuring Astrocyte Phagocytic Activity
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Phagocytic activity was measured with a pHrodo Green E. coli BioParticles conjugate (catalog number P35366; Life Technologies). The particles are conjugated to pHrodo dye, the fluorescence of which increases in an acidic environment such as that of the phagosome. For measurement of phagocytic activity, astrocytes were plated onto 24-well culture plates for 3 days and incubated with pHrodo Green E. coli BioParticles (Invitrogen, P35366) for 4 hours. Phagocytic activity was determined based on the fluorescence intensity of the cells by confocal microscopy.
Phagocytic activity was also measured by spectrophotometry. Plate astrocytes into a 96-well plate as experimental wells. Leave two wells empty of cells for every control well, so that a no-cell control background subtraction may be performed. Astrocytes were cultured for 24 h, and the culture medium was then replaced with 200 μL of pHrodo Green E. coli BioParticles conjugate solution in experimental and no-cell control wells. Cover and transfer the microplate to an incubator warmed to 37°C for 4 hours to allow phagocytosis and acidification to reach its maximum. Scan all experimental and no-cell control wells of the microplate in the fluorescence plate reader (BioTek Synergy H1 instrument). Phagocytic activity was determined based on relative fluorescence intensity of pHrodo particles in astrocytes.
Phagocytic activity was also measured by spectrophotometry. Plate astrocytes into a 96-well plate as experimental wells. Leave two wells empty of cells for every control well, so that a no-cell control background subtraction may be performed. Astrocytes were cultured for 24 h, and the culture medium was then replaced with 200 μL of pHrodo Green E. coli BioParticles conjugate solution in experimental and no-cell control wells. Cover and transfer the microplate to an incubator warmed to 37°C for 4 hours to allow phagocytosis and acidification to reach its maximum. Scan all experimental and no-cell control wells of the microplate in the fluorescence plate reader (BioTek Synergy H1 instrument). Phagocytic activity was determined based on relative fluorescence intensity of pHrodo particles in astrocytes.
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