We seeded GECs on 24-well plates and stimulated them for 24 h. Then we changed the medium and put them on collagen-coated transwells with podocyte monolayers for 24 h (Corning Costar Corp., Cambridge, MA, USA). After stimulation, we put transwells in new plates, and we measured cytotoxicity, cell polarity, and permeability to albumin. For cytotoxicity, we used 250 μg/mL XTT (Sigma Aldrich) solution. Supernatants and filtrates were collected after 2 hr. and analyzed at a wavelength of 450 nm. Cell polarity was analyzed by measuring trans-epithelial electrical resistance (TEER) with an epithelial volt-ohm meter (EVOM, World Precision Instruments, Inc., Sarasota, FL, USA). We also evaluated permeability to albumin by diffusion of Trypan blue-albumin complexes across transwells. Aliquots of the medium from the upper and the lower wells were transferred to a 96-well plate and analyzed at the 590 nm wavelength (Model 680 Spectrophotometer, Biorad, Hercules, CA, USA). Results are expressed as arbitrary units (upper medium O.D./lower medium O.D.).
Model 680 spectrophotometer
Manufactured by Bio-Rad
Sourced in United States, Japan
The Model 680 spectrophotometer from Bio-Rad is a compact and versatile instrument designed for accurate absorbance measurements. It utilizes a monochromator to select specific wavelengths of light, allowing for quantitative analysis of samples across a wide range of applications.
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5 protocols using model 680 spectrophotometer
1
Assessing Podocyte Barrier Functions
2
Quantification of IP3 levels
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The levels of IP3 were determined using an IP3 ELISA kit (Cusabio Biotech Co., Ltd, Wuhan, China), according to the manufacturer’s instructions. The ASMC culture medium was removed and the cells were incubated with 0.1 mmol/l HClO4 for 20 min. The cells were centrifuged at 170 × g for 15 min at room temperature and the supernatant was collected for analysis. An anti-IP3 detection antibody was added and incubated at 37°C for 60 min, followed by the addition of substrate solution for 15 min at 37°C. The reaction was terminated following the addition of stop solution and the plates were read at an absorbance of 450 nm using a Model 680 spectrophotometer (Bio-Rad Laboratories, Inc.). The effect of formoterol on the expression of IP3 was determined using the following formula: Inhibition of ACh-induced IP3 accumulation (%) = (IP3 levels in the control group - IP3 levels in the treatment group) / IP3 levels in the control group × 100%.
3
Quantifying IP3 Levels in ASMC
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The levels of IP3 were determined using an IP3 ELISA kit according to the manufacturer’s instructions. Briefly, the ASMC culture medium was removed and the cells were incubated with 0.1 mmol/l HClO4 for 20 min. The cells were centrifuged at 170 × g for 15 min at room temperature, and the supernatant was collected for analysis. An anti-IP3 detection antibody was added and incubated at 37 °C for 60 min, followed by the addition of substrate solution for 15 min at 37 °C. The reaction was terminated following the addition of stop solution and the plates were read at an absorbance of 450 nm to test the optical density (OD) value and calculate the IP3 concentration using a Model 680 spectrophotometer (Bio-Rad Laboratories, Inc.). The effect of ICI118,551 on the expression of IP3 was determined by the following formula: Inhibition of Ach-induced IP3 accumulation (%) = (IP3 levels in the control group − IP3 levels in the treatment group)/IP3 levels in the control group × 100%.
4
Podocyte Permeability and Cytotoxicity Assay
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We seeded GECs on 24-well plates and stimulated them for 24 h. Then we changed the medium and put them on collagen-coated transwells with podocyte monolayers for 24 h (Corning Costar Corp., Cambridge, MA). After stimulation, we put transwells in new plates, and we measured cytotoxicity, cell polarity, and permeability to albumin. For cytotoxicity, we put 250 μg/mL XTT (Sigma Aldrich) solution on podocytes. Supernatants and filtrates were collected after two h and analyzed at a wavelength of 450 nm. Cell polarity was analyzed by measuring transepithelial electrical resistance (TEER) with an epithelial volt-ohm meter (EVOM, World Precision Instruments, Inc., Sarasota, FL, USA). We also evaluated permeability to albumin by diffusion of Trypan bluealbumin complexes across transwells. Aliquots of the medium from the upper and the lower wells were transferred to a 96-well plate and analyzed at the 590 nm wavelength (Model 680 Spectrophotometer, Biorad, Hercules, CA). Results are expressed as arbitrary units (upper medium O.D./lower medium O.D.).
5
DPPH Antioxidant Activity Measurement
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The method for measuring anti-oxidant power by DPPH is based on the ability of a compound to reduce the DPPH ° radical. The scavenging of free radicals of the lyophilized aqueous extract and the powder of Lannea microcarpa was evaluated according to the method of Kim [11] . Twenty (20) µL of different concentrations of aqueous or reference extracts (Trolox) were mixed with 200 µL of a methanolic solution of DPPH (0.08 mg / mL) in the wells of a 96-well microplate. The absorbance was read at 490 nm with the BioRad Model 680 spectrophotometer (Japan) after 30 min incubation at room temperature. Each test was performed in triplicate, and the blank was the diluting solvent of the extract or standard. The curve of inhibition of absorbance versus concentration of extract or Trolox was established to determine the 50% inhibitory concentration (CI 50 ).
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