The intracellular accumulation of reactive oxygen species (ROS) in the RAW264.7 macrophages was determined with carboxy-2’,7’-dichloro-dihydro-fluorescein diacetate (DCFHDA) method.[15 (link)] Cells RAW264.7 were plated in 35-mm culture dishes at 5.0 × /104 cells/cm2. Then, the medium is changed with a new one. Cells seeded on 96-well/plates were incubated with DCFHDA probe for 40 min. At the end of this period, medium was removed and cells exposed to the extract under investigation at a concentration to 50, 100, and 200 μg/ml. After incubating exposed cells at 37°C for 24 h, fluorescence was measured at 488 nm (excitation) and 535 nm (emission) wavelengths on a microplate reader (Molecular Devices Spectra MAX Gemini X).
Spectra max gemini x
Manufactured by Molecular Devices
The Spectra MAX Gemini X is a multi-mode microplate reader that can perform absorbance, fluorescence, and luminescence measurements. It is designed to provide accurate and reliable data for a wide range of applications in life science research and drug discovery.
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4 protocols using spectra max gemini x
1
Quantifying Intracellular ROS in RAW264.7 Cells
2
Senescence-Associated β-Galactosidase Assay
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Senescence-associated β-galactosidase (SA-β-Gal) was performed using the fluorescent cellular senescence assay kit from Cell Biolabs (San Diego, CA, USA) and following the manufacturer’s recommended protocol. Briefly, cells were washed with cold PBS and lysed. Lysates were centrifuged and supernatant was collected. After transfer to fluorescence 96-well plates, the lysates were incubated with the assay buffer for 1 h at 37 °C. The reaction was stopped, and the fluorescence was measured at 360 (excitation) and 465 nm (emission) on a dark microplate reader (Molecular Devices Spectra MAX Gemini X).
3
ROS Measurement via DCFH-DA Assay
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ROS measurement was performed as described previously [28 (link)]. Cells (5 × 105) were resuspended with carboxy-2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA Sigma, St Louis, MO, USA) probe and incubated at 37 °C for 30 min in the dark. The fluorescence was determined after 24 h of treatment measuring the absorbance at 485 (excitation) and 535 nm (emission) wavelengths using a dark microplate reader (Molecular Devices Spectra MAX Gemini X).
4
Intracellular ROS Accumulation in H9c2 Cells
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The intracellular accumulation of ROS in the H9c2 rat cardiomyoblast cell line was determined with carboxy-2′,7′-dichloro-dihydro-fluorescein diacetate (DCFHDA) method [14 (link)]. Cells H9c2 rat heart-derived embryonic myocytes are obtained from American Type Culture Collection, Manassas, VA, USA (CRL-1446), which was cultured with DMEM/F12 supplemented with 100 U/mL penicillin G, 10% (v/v) foetal bovine serum, 2 mM/L-glutamine, and 100 mg/mL streptomycin. Cells were incubated using 5% CO2 and 95% air at 37°C, after the cells were plated in 35-mm culture dishes at 5.0 × 10−4 cells/cm2. Then, the medium is changed with a new one. Cells seeded on 96-well plates were incubated with DCFHDA probe for 40 min. At the end of this period, medium was removed and cells were exposed to the flavonoids under investigation at a concentration of 10, 50, and 100 μg/mL. After incubating exposed cells at 37°C for 24 h, fluorescence was measured at 488 nm (excitation) and 535 nm (emission) wavelengths on a microplate reader (Molecular Devices Spectra MAX Gemini X).
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