Oil Red O staining was used to confirm the mature 3T3-L1 adipocytes. In brief, the mature 3T3-L1 cells were fixed with ice-cold acetone for 30 min and then stained with 0.3% Oil Red O (Sigma–Aldrich, Darmstadt, Germany)/60% isopropanol solution (Invitrogen) for 2 h. The stained lipid droplets were observed under phase contrast microscopy (Nikon Corporation, Tokyo, Japan).
Phase contrast microscopy
Manufactured by Nikon
Sourced in Japan, United States
Phase-contrast microscopy is an optical imaging technique that enhances the contrast of transparent samples, allowing for the visualization of fine details that would otherwise be difficult to observe. This technique uses specialized optical components to convert small differences in refractive index within the sample into variations in image brightness, making it particularly useful for studying living cells and other transparent specimens.
Automatically generated - may contain errors
Lab products found in correlation
Alizarin red s solution, by Merck Group (3 mentions)
Formaldehyde, by Merck Group (3 mentions)
Matrigel, by BD (3 mentions)
Glutaraldehyde, by Merck Group (2 mentions)
Image pro plus 6, by Nikon (2 mentions)
Oil red o, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Laminin, by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor (fgf), by Thermo Fisher Scientific (1 mentions)
Human epidermal growth factor (hegf), by Thermo Fisher Scientific (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Methylcellulose, by Merck Group (1 mentions)
40 micron, by Corning (1 mentions)
Scion image program, by Techcomp Instruments (1 mentions)
Hemocytometer, by Marienfeld (1 mentions)
Incubator shaker, by Eppendorf (1 mentions)
Senescence associated β galactosidase staining kit, by Beyotime (1 mentions)
45 protocols using phase contrast microscopy
1
Oil Red O Staining of Mature 3T3-L1 Adipocytes
2
Adhesion of RPE Cells to ECM Proteins
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Glass coverslips (13 mm, acid-washed) were coated with collagen I, collagen IV, fibronectin or laminin (1 μg/ml, Sigma) for at least 2 h at RT. The coverslips were then washed twice with sterile PBS. Cultured RPE cells were briefly trypsinized (~3 min at 37°C), pelleted, washed and resuspended in Miller medium to a final concentration of 100,000 cells/ml. 500 μl (28,000 cells/cm2) of this solution were added to each coverslip in a well of a 24-well-plate. The plates were then incubated in a shaking incubator (Luckham R300) at 10 rounds per minute at 37°C for 1 h. After the incubation, the coverslips were washed three times with PBS to wash away loose cells. The attached cells were then visualized and counted under phase contrast microscopy (Nikon). Five random fields (at left, right, middle, top and bottom of coverslip) were chosen from each coverslip and the number of attached cells was counted. The average number of cells adhering was counted and normalized to the average number of attached cells under control conditions (non-coated glass coverslip). Each condition contained three coverslips and experiments were repeated three times. All data was analyzed using one-way ANOVA with Dunnett’s post hoc test using GraphPad Prism software. The results are presented as mean + SEM. Significance values were represented as: **P < 0.01.
3
Culturing Primary Rat Hippocampal Neurons
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Primary cultured hippocampal neurons were prepared from the rat neonates as described previously (Banker et al., 1977). Briefly, the hippocampi were dissected out and trypsinized (0.25% bovine trypsin) for 9–10 minutes at 37°C. The cells were plated in Neurobasal™ Medium with 2% B27 supplement, 10 μL/mL penicillin-streptomycin, 1% GlutaMAX, 0.4% BSA and 20 mM HEPES at 37°C in a humidified 5% CO2 incubator. Neurons were characterized using phase contrast microscopy (Nikon Corporation, Tokyo, Japan) and immunocytochemistry for GAP-43. Neuronal cultures were over 90% pure.
4
Assessing Estrous Cycle in PCOS Mice
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On approximately the 20th day after the first treatment to establish the PCOS mice model, the estrous cycle of the mice was evaluated by vaginal cytology for 10 consecutive days. Mouse secretions were obtained from the vagina by using a cotton swab dipped in PBS, then spread evenly on the slide. Meanwhile cell morphology and estrous cycles were observed by phase-contrast microscopy (Nikon, Japan).
5
Mammosphere Formation and Passage Assay
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As reported earlier [53 (link)], cells were cultured in 96-well Ultra-Low attachment plates (250 cells/well) with serum-free medium supplemented with 1 × B27 Supplement (Thermo Fisher Scientific, Sunnyvale, CA, USA), 1 × 2-Mercaptoethanol, 5 μg/ml insulin (Thermo), 1 μg/ml hydrocortisone (Sigma-Aldrich, Saint-Louis, MO, USA), 20 ng/ml human epidermal growth factor (Thermo), 10 ng/ml fibroblast growth factor (Thermo) and 0.5% Methyl cellulose (Sigma-Aldrich). TrypLE Express (Thermo) was used for passaging mammospheres.
In mammosphere formation assay, mammospheres were cultured in the presence of B591 for 5 days as described above and then were counted and imaged under a phase-contrast microscopy (Nikon, Japan).
In mammosphere passage assay, mammospheres were cultured as described above and treated with B591 in the first generation and passaged two additional generations without B591 treatment. For passaging, mammospheres were collected by centrifuge (1000 g, 5 min, 4℃) and dissociated by TrypLE Express (Thermo), then the mixture was passed through a cell strainer (40-micron, Corning) to obtain the single cell suspension for subsequently mammosphere formation as described above.
In mammosphere formation assay, mammospheres were cultured in the presence of B591 for 5 days as described above and then were counted and imaged under a phase-contrast microscopy (Nikon, Japan).
In mammosphere passage assay, mammospheres were cultured as described above and treated with B591 in the first generation and passaged two additional generations without B591 treatment. For passaging, mammospheres were collected by centrifuge (1000 g, 5 min, 4℃) and dissociated by TrypLE Express (Thermo), then the mixture was passed through a cell strainer (40-micron, Corning) to obtain the single cell suspension for subsequently mammosphere formation as described above.
6
Quantifying Osteogenic Differentiation by ARS
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For measurement of bone nodule formation after osteogenic differentiation, detection of the extracellular matrix calcium deposits was determined using ARS staining. Briefly, after culturing with osteogenic medium for 14 days, the cells were washed and fixed routinely, the extracellular matrix calcium deposits were measured by staining the cells with 40 mM alizarin red S solution (Sigma-Aldrich). The cells and nodule formation were captured by phase-contrast microscopy (Nikon, Japan). The density was analyzed by Image-Pro Plus 6.0.
7
Wound Healing Assay with Additives
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NIH 3T3 cells were seeded in a tissue culture 6-well plate at an initial density of 2.4 × 105 cells/cm2 overnight. A micropipette tip was used to create a wound in the monolayer by scraping. A total of 10 (%, w/v) RB maltodextrin were added in each treatment well with or without an addition of 100 ppm of various additives including aloe vera, curcumin, hydroxyproline, ascorbic acid, L-arginine, lactic acid, and kojic acid, which were added separately to each well. Another two wells were treated with Multidex and media only (control), respectively. Wound closure was observed by phase-contrast microscopy (NIKON, Japan) and digital images were taken at the interval time of 3 h up to 24 h.
8
Wound Healing Assay with HaCaT Cells
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Human keratinocyte cell line (HaCaT) was seeded in a tissue culture 6-well plate at an initial density of 3 × 104 cells/well overnight. When cells reached 95% confluence, a micropipette tip was used to scratch a wound on the monolayer as described previously26 (link). Wells were then treated with H2O2 and/or DeinoPol (10 μg), and wound closure was observed by phase contrast microscopy (Nikon; Tokyo, Japan). Digital images were taken at 18 h.
9
Measuring Cell Motility Assays
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Cell motility was assessed by the wound-healing and phagokinetic gold sol assays as previously described [57 (link)]. For phagokinetic gold sol assay, cells were plated and treated or not with piperine and TGF-β1 as mentioned. After treatment, cells were detached, and 5.0 × 102 cells were plated onto gold sol-coated well and incubated for 18 h. Photographs were taken by phase-contrast microscopy (Nikon) at 80 × magnification; the track area of 200 cells was measured by using the Scion Image program, and expressed as squared pixels. For the wound-healing assay, cells were treated as above and scratches were made on the cell monolayers with plastic pipette tips by moving them perpendicularly to the lines marked at the bottom of wells. The cells were then rinsed and incubated in culture medium for 18 h. Pictures were taken at 0 h and 18 h. The procedure was based on previous studies [57 (link),58 (link)].
10
Alginate Hydrogel Coating Technique
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In preparation for coating, the PBAA was first dissolved in a mannitol buffer containing 300 mM d -mannitol and 25 mM Hepes to a concentration of 0.3 wt %, and the pH was adjusted to around 7.2 using 6 M HCl. The final coating solution was diluted to a final concentration of 0.01 wt % with the mannitol buffer before sterile filtering.
PBAA coating immediately followed the preparation of gelled samples. Samples were first incubated for 5 min in a Hepes buffer [25 mM Hepes, 1.2 mM MgCl2 × 6H2O, 4.7 mM KCl, and 132 mM NaCl2 (pH 7.4)] to destabilize the alginate crosslinks. Samples were then rinsed twice in the mannitol buffer for 2 min each. The samples were then incubated in the PBAA coating solution for 15 to 20 min with mild agitation. The process was monitored by phase contrast microscopy (Nikon), through which the formation of a thin coating could be observed. Coated samples were then rinsed three times with normal saline before culturing in media or storage in saline supplemented with 2 mM CaCl2, in the case of acellular samples.
PBAA coating immediately followed the preparation of gelled samples. Samples were first incubated for 5 min in a Hepes buffer [25 mM Hepes, 1.2 mM MgCl2 × 6H2O, 4.7 mM KCl, and 132 mM NaCl2 (pH 7.4)] to destabilize the alginate crosslinks. Samples were then rinsed twice in the mannitol buffer for 2 min each. The samples were then incubated in the PBAA coating solution for 15 to 20 min with mild agitation. The process was monitored by phase contrast microscopy (Nikon), through which the formation of a thin coating could be observed. Coated samples were then rinsed three times with normal saline before culturing in media or storage in saline supplemented with 2 mM CaCl2, in the case of acellular samples.
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