Intracellular ROS levels were measured by staining with the cell permeant reagent, 2′,7′-dichlorofluorescein diacetate (DCF-DA) (Sigma-Aldrich Co.). On attaining 70–80% confluence, RAW264.7 cells were exposed to vehicle or two different concentrations of MCE, precultured for 2 h in a 37 °C incubator, followed by LPS stimulation (1 μg/mL) for 24 h. The cells were then incubated with 100 μM DCF-DA for 15 min at 37 °C. After washing with 1× PBS, the resultant green fluorescence was observed at 200× magnification using a fluorescent microscope (Eclipse TX100, Nikon, Tokyo, Japan). The cell morphology was also observed under a light microscope (Leica Microsystems, Heerbrugg, Switzerland) at 200× magnification.
Eclipse tx100
Manufactured by Nikon
Sourced in Japan
The Eclipse TX100 is a microscope designed for routine observation and documentation of samples. It features a built-in camera port and supports a range of objectives to accommodate various specimen types. The Eclipse TX100 provides a reliable and versatile solution for basic laboratory tasks.
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9 protocols using eclipse tx100
1
Measuring Intracellular ROS in RAW264.7 Cells
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Intracellular ROS Measurement in HepG2 Cells
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Intracellular reactive oxygen species (ROS) levels were measured by staining with 2’,7’-dichlorofluorescein diacetate (DCFH-DA) (Cat. No. D6883, Sigma-Aldrich Co.), which is a cell permeable and nonfluorescent agent that can be deacetylated by intracellular esterases to nonfluorescent DCFH. In the presence of ROS, DCFH was converted to highly fluorescent DCF intracellularly. Briefly, HepG2 cells were seeded at a density of 5 × 105 cells/2 ml in 6-well plates, then grown with four different concentrations of GEGR for 24 h in a 37°C incubator. After washing once with ×1 PBS, the cells were incubated with 5 μM H2O2 for another 12 h. Next, cells were incubated with 25 μM DCFH-DA for 30 min at 37°C. The cells were then washed twice with PBS, after which the green fluorescence was observed using a fluorescent microscope (Eclipse TX100, Nikon, Tokyo, Japan).
3
Quantifying Proliferating Cells Using PCNA
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PACs were seeded in chamber slides and cultured in standard conditions until reaching a confluence of 70%. Proliferating cells were stained with anti-mouse PCNA antibody and PCNA-positive cells were counted using the fluorescence microscope (Nikon Eclipse TX-100).
4
Measuring Intracellular ROS in RAW264.7 Cells
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Intracellular ROS levels in RAW264.7 cells were measured by staining with 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich Co.), which is a cell permeable, nonfluorescent agent that can be deacetylated by intracellular esterases to form nonfluorescent DCFH. In the presence of ROS, DCFH is converted to highly fluorescent DCF intracellularly. Briefly, RAW364.7 cells were seeded at 5×105 cells/2 ml in 6-well plates, then grown with two different concentrations of BAW for 2 h in a 37°C incubator. After washing once with 1× PBS, the cells were incubated with 1 µg/ml of LPS for another 24 h. Next, cells were incubated with 25 µM DCFH-DA for 30 min at 37°C. Finally, the cells were washed twice with PBS, after which the green fluorescence was observed at 200× magnification via fluorescence microscopy (Eclipse TX100, Nikon, Tokyo, Japan).
5
Quantifying Intracellular and Tissue ROS Levels
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Intracellular ROS levels were measured in B35 cells (neural neuroblast cells that originate from the neuroblastoma of BDIX rats) by staining with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich Co.), as previously described [22 (link)]. Briefly, B35 cells were seeded at 5 × 105 cells/2 mL in six-well plates and incubated with 100 μg/mL of GEGR for 1 h at 37 °C. After washing with 1× PBS, the cells were incubated with H2O2 (100 μM; Junsei Chemical Co.) for 24 h. Next, the stimulated cells along with 25 μM DCFH-DA were incubated for 30 min at 37 °C. Finally, the cells were washed twice with 1× PBS, and the presence of green fluorescence was observed under a fluorescence microscope (200× magnification; Eclipse TX100, Nikon, Tokyo, Japan).
ROS level in the brain cortex tissue was assessed by employing a proprietary quenched fluorogenic probe and the reagents provided in the OxiSelect™ In Vitro ROS/RNS Assay Kit (Cell Biolabs Inc., San Diego, CA, USA). Briefly, the brain cortex tissue samples (100 mg) were homogenized in 600 μL of 1× PBS using a glass homogenizer. Each sample and standards (50 μL) were mixed with Catalyst (50 μL) in 96-well plate for 5 min. DCFH solution (100 μL) were added into this mixture and incubated under the light protection for 15–45 min at room temperature. The final fluorescence of each well were measured at 480 nm excitation/530 nm emission.
ROS level in the brain cortex tissue was assessed by employing a proprietary quenched fluorogenic probe and the reagents provided in the OxiSelect™ In Vitro ROS/RNS Assay Kit (Cell Biolabs Inc., San Diego, CA, USA). Briefly, the brain cortex tissue samples (100 mg) were homogenized in 600 μL of 1× PBS using a glass homogenizer. Each sample and standards (50 μL) were mixed with Catalyst (50 μL) in 96-well plate for 5 min. DCFH solution (100 μL) were added into this mixture and incubated under the light protection for 15–45 min at room temperature. The final fluorescence of each well were measured at 480 nm excitation/530 nm emission.
6
Intracellular ROS Measurement in RAW264.7 Cells
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The intracellular ROS levels in RAW264.7 cells were evaluated using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) fluorescent probe. The cell permeable DCFH-DA was deacetylated using intracellular esterases to form non-fluorescent 2′,7′-dichlorodihydrofluorescein (DCFH). In the presence of ROS, the DCFH was intracellularly converted to the highly fluorescent 2′,7′-dichlorofluorescein (DCF). Briefly, six-well plates were seeded with RAW364.7 cells at a density of 5 × 105 cells/2 mL, then cultured in the presence of two concentrations of BAW for 2 h in a 37 °C incubator. After washing with 1× PBS, the cells were treated with either vehicle (DMSO) or BAW (100 µg/mL) for 2 h, exposed to LPS (1 μg/mL) for another 24 h, then incubated with 25 μM DCFH-DA for 30 min at 37 °C. Finally, the cells were observed at 200× magnification for green fluorescence using a fluorescence microscope (Eclipse TX100, Nikon, Tokyo, Japan).
7
Immunofluorescent Detection of p53 in Prostate Tissues
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For detection of p53 protein via IF staining analysis, prostate tissues of subset groups were fixed in 10% formalin for 48 h, embedded in paraffin block, sliced into 4 µm thick sections, and mounted on a glass slide. Sections were then deparaffinized with xylene, rehydrated with different concentrations of EtOH, and pretreated with blocking buffer containing 10% goat serum in 1× PBS solution, for 30 min at room temperature. The pretreated sections were subsequently incubated with anti-p53 antibody (Sigma-Aldrich Co.), diluted 1:300 in blocking buffer. After thorough washing in 1× PBS solution, the slide sections were incubated with goat FITC-labeled anti-rabbit IgG serum for 1 h, washed thrice in 1× PBS for 3 min each, and mounted with vector shield mounting medium. Finally, the green fluorescence in stained cells was observed at 400× magnification via fluorescence microscopy (Eclipse TX100, Nikon, Tokyo, Japan).
8
Measurement of Intracellular ROS in NHDF Cells
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Intracellular ROS levels were measured in NHDF cells by staining with 2′,7′-dichlorofluorescein diacetate (DCF-DA) (Sigma-Aldrich Co.) as previously described [41 (link)]. Briefly, NHDF cells seeded at 4.5 × 105 cells/3 mL in 6-well plates were cultured to 70–80% confluent, irradiated at 50 mJ, and treated with CPO or GEGR and 25 μM DCFH-DA and incubated for 30 min at 37 °C. After washing twice with 1× PBS, the degree of green fluorescence was observed in DCF-DA stained cell under a fluorescence microscope (200×; Eclipse TX100, Nikon, Tokyo, Japan).
9
Measuring ROS Levels in RAW264.7 Cells
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ROS levels were measured by staining with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich; Merck KGaA). Briefly, RAW264.7 cells were seeded at a density of 1×105 cells/ml in 24-well plates for 24 h, and exposed to three different concentrations of EMfC (100, 200 or 400 µg/ml) or DMSO for 2 h in a 37°C incubator. The cells were subsequently stimulated with 1 µg/ml LPS for further 24 h. Cells were then incubated with 25 µM DCFH-DA for 30 min at 37°C, washed twice with PBS, and observed for green fluorescence at ×200 magnification using a fluorescent microscope (Eclipse TX100; Nikon).
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