After treating 3T3-L1 cells for 12 days, the cells were carefully rinsed with PBS and fixed with 4% formaldehyde at room temperature for 1 h, followed by washing with 60% isopropanol. The 3T3-L1 cells were then stained with Oil Red O solution (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min and washed with 60% isopropanol and distilled water. Stained lipid droplets were quantified at an optical density of 492 nm using a microtiter plate reader (Allsheng, Hangzhou, China). For the quantification of cellular triglycerides, a triglyceride assay kit (Abcam, Cambridge, UK) was used. Briefly, after treating 3T3-L1 cells with BLs (0, 5, 10, or 20 μg/mL), the cells were washed with PBS and resuspended in 5% NP-40, followed by boiling for 5 min. The cell suspension was centrifuged at 13,000× g for 2 min and the supernatants were mixed with lipases to convert triglycerides into glycerol. After adding a triglyceride probe and a triglyceride enzyme mix, the mixture was incubated at room temperature for 1 h and the optical density was measured at 570 nm using a microtiter plate reader (Allsheng). The concentration of triglycerides was determined based on the standard curve of glycerol.
Microtiter plate reader
Manufactured by Allsheng
Sourced in China
The Microtiter plate reader is a versatile laboratory instrument used to detect and quantify various analytes in microplate format. It is designed to measure absorbance, fluorescence, or luminescence signals from samples contained in the wells of a microtiter plate. The device is commonly used in applications such as enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and other microplate-based experiments.
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5 protocols using microtiter plate reader
1
Quantification of Lipid Accumulation in 3T3-L1 Cells
2
Cell Proliferation, Colony Formation, and Migration Assays
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The cells were seeded in 96‐well plates at 1,000 cells per well for the cell proliferation assays. Cell proliferation was evaluated using 10% CCK‐8 (SolarBio) diluted in standard culture media for 2 h. Proliferation rates were determined at 0, 24, 48, 72, and 96 h after seeding, and quantification was performed on a microtiter plate reader (ALLSHENG, Hangzhou, Zhejiang, China) measured at UV wavelength (λ) = 450 nm.
For colony formation assay, cells were seeded in 6‐well plates at a density of 500 cells/well. The cells were then cultured in complete media for 4‐6 weeks until the size of a single colon reached 2‐5 mm. Then the colonies were fixed by 4% paraformaldehyde and stained by 1% leucocrystal violet for 5 minutes.
For the cell migration and invasion transwell assays, 50,000 cells in 400 μL media with 10% fetal bovine serum were plated on the top chambers of Transwell Clear Polyester Membrane Inserts (for the migration assay, Corning Costar, New York, USA) and BioCoat Matrigel Invasion Chambers (for the invasion assay, Corning Costar), while culture media with 10% FBS was applied to the bottom. After 16‐72 hours, migrated or invaded cells were stained with crystal violet and counted under a microscope (Olympus, Tokyo, Japan).
For colony formation assay, cells were seeded in 6‐well plates at a density of 500 cells/well. The cells were then cultured in complete media for 4‐6 weeks until the size of a single colon reached 2‐5 mm. Then the colonies were fixed by 4% paraformaldehyde and stained by 1% leucocrystal violet for 5 minutes.
For the cell migration and invasion transwell assays, 50,000 cells in 400 μL media with 10% fetal bovine serum were plated on the top chambers of Transwell Clear Polyester Membrane Inserts (for the migration assay, Corning Costar, New York, USA) and BioCoat Matrigel Invasion Chambers (for the invasion assay, Corning Costar), while culture media with 10% FBS was applied to the bottom. After 16‐72 hours, migrated or invaded cells were stained with crystal violet and counted under a microscope (Olympus, Tokyo, Japan).
3
Cell Proliferation Assay in 96-Well
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Cells were seeded in 96-well plates at 2000 cells/well for the CCK-8 assay. After 0, 24, 48, 72, and 96 h of planting, relative cell numbers were evaluated using 10% CCK-8 (SolarBio, Beijing, China) diluted in standard culture media for 2 h. Quantification was performed on a microtiter plate reader (ALLSHENG, Hangzhou, Zhejiang, China) at a UV wavelength (λ) of 450 nm.
4
RAW 264.7 and PDL Cells Cytokine Response
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The RAW 264.7 cells (5 × 10 5 cells/mL) or PDL cells (2.5 × 10 5 cells/mL) were plated onto 48-well culture plates and treated with various concentrations of M157-W (0, 0.1, 1, 10, or 100 μg/mL or 100 μg/mL, respectively) or unfermented whey (100 μg/mL) in the presence or absence of P. gingivalis LPS (1 μg/mL) for the indicated time periods. Subsequently, culture supernatants were collected and IL-1β, IL-6, and IL-8 secretions were determined using a commercial ELISA kit (R&D Systems) according to the manufacturer's instructions. To determine nitric oxide production, the culture supernatants were mixed with the same volume of the Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2% phosphoric acid) and incubated at room temperature for 5 min. Then, the optical density was measured at 540 nm using a microtiter plate reader (Allsheng) with NaNO 2 as the standard.
5
Inhibition of S. mutans Biofilm Formation
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The overnight culture of S. mutans was diluted to 1 × 10 7 cfu/mL in BHI broth containing 0.05% sucrose, and 100 μL of S. mutans suspension was then transferred to 96-well microtiter plates in various concentrations of M157-W or unfermented whey (0, 0.001, 0.01, 0.1 and 1 mg/mL; 100 μL). To evaluate the inhibitory effect of M157-W on S. mutans biofilm, the bacteria were incubated at 37°C for 24 h. Subsequently, the bacteria were gently washed with PBS to remove unattached bacteria and then stained with 0.1% crystal violet for 30 min. After excess stain was removed by gently washing with PBS, the stained S. mutans biofilm was dissolved in 0.1% acetic acid and 95% ethanol and its absorbance was measured at 595 nm using a microtiter plate reader (Allsheng).
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