Cells at 80% confluence were washed twice with PBS and fixed with 4% formaldehyde for 5 min at room temperature. Subsequently, cells were incubated at 37°C with β-gal staining solution (Beyotime Institute of Biotechnology) overnight according to the manufacturer's protocol. Stained cells were visualized using an inverted phase contrast light microscope (Olympus Corporation). In total, >300 random cells were analyzed per sample, and percentages of stained cells were calculated.
Inverted phase contrast light microscope
Manufactured by Olympus
Sourced in Japan
The Inverted phase-contrast light microscope is a laboratory equipment designed to observe and analyze samples. It utilizes phase-contrast illumination to enhance the visibility of transparent specimens. The instrument's core function is to magnify and provide a detailed visual representation of the sample under examination.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (4 mentions)
Mitomycin c, by Merck Group (3 mentions)
Dp controller software, by Olympus (2 mentions)
Stemxvivo serum free media, by R&D Systems (2 mentions)
Ultra low attachment 6 well plate, by Corning (2 mentions)
β gal staining solution, by Beyotime (1 mentions)
Dapi dye, by Olympus (1 mentions)
Pkh67, by Merck Group (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Transwell filter, by Merck Group (1 mentions)
Hydrocortisone, by Bio-Techne (1 mentions)
Heparin, by Bio-Techne (1 mentions)
Recombinant human il 6, by Thermo Fisher Scientific (1 mentions)
G8450, by Solarbio (1 mentions)
H8060, by Solarbio (1 mentions)
Matrigel, by Olympus (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Crystal violet, by Thermo Fisher Scientific (1 mentions)
19 protocols using inverted phase contrast light microscope
1
Senescence-Associated β-Galactosidase Assay
2
Exosome-mediated Fibroblast Proliferation Regulation
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HSFBs were precultured in 6-well plates (1×104 cells/well) in DMEM containing 10% FBS and 1% (v/v) penicillin/streptomycin for 4 h. Then, 20 µg miR-29a-modified hADSCs-exo (mimics-exo or inhibitor-exo) was added to the culture medium of HSFBs. An equivalent volume of exosome diluent PBS was added as the control group. After the exosomes were added, the cells were incubated at 37°C with 5% CO2 and imaged at 0, 24 and 48 h using an inverted-phase contrast light microscope (Olympus Corporation; magnification, ×10) to observe the proliferation of the cells.
In addition, whether HSFBs can directly take up in hADSCs-exo was detected via immunofluorescence labeling. Exosomes were labeled with the red fluorescent linker PKH67 (Sigma-Aldrich; Merck KGaA), as previously reported (37 (link)). HSFBs were treated with PKH67-labeled exosomes for 24 h at 37°C with 5% CO2. DAPI dye (cat. no. ab104139; Abcam) was used for nuclear staining for 10 min at room temperature. After washing by distilled water to remove the uninternalized exosomes and excess DAPI dye, the fluorescent images were visualized using a confocal microscope (Olympus Corporation; magnification, ×40).
In addition, whether HSFBs can directly take up in hADSCs-exo was detected via immunofluorescence labeling. Exosomes were labeled with the red fluorescent linker PKH67 (Sigma-Aldrich; Merck KGaA), as previously reported (37 (link)). HSFBs were treated with PKH67-labeled exosomes for 24 h at 37°C with 5% CO2. DAPI dye (cat. no. ab104139; Abcam) was used for nuclear staining for 10 min at room temperature. After washing by distilled water to remove the uninternalized exosomes and excess DAPI dye, the fluorescent images were visualized using a confocal microscope (Olympus Corporation; magnification, ×40).
3
Quantitative Wound Healing and Cell Migration Assays
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Wound healing and Transwell assays were performed as previously described [4 (link), 11 (link)]. Briefly, after appropriate treatments, cells were seeded in 6-well plates and cultured until they reached 90% confluence. A 10-μl micropipette tip was used to make a wound. Cells were monitored at 0 h and 48 h after scratching and images of wound healing were captured (magnification of 100×) using a inverted phase contrast light microscope (Olympus, Tokyo, Japan) with DP Controller software (Olympus Life Science, Tokyo, Japan). Cell migration was quantified by measuring the wound healing index; i.e., the wound area healed by the cells at 48 h after scratching relative to the wound area at 0 h, using ImageJ software. For the Transwell migration or invasion assays, cells were resuspended in DMEM without serum and seeded into the upper chamber of 8-μm Transwell filters (Merck Millipore, Berlin, Germany). The invasion assay was performed using filters pre-coated in 1:3 diluted matrigel (BD Biosciences, Bedford, MA, USA) while the migration assay was not. DMEM containing 15% FBS was added to the lower chambers (24-well plate) and the cells were incubated 16 h for the migration assay and 24 h for the invasion assay. The invaded or migrated cells were quantified after 0.1% crystal violet staining in five randomly selected fields (magnification of 200×).
4
Tumorsphere Formation Assay
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Single cell was plated in ultralow attachment 6-well plates (Corning) at 25,000 cells per well for primary tumorsphere formation. After incubation for 7 days, mammospheres were collected and dissociated by trypsin. As the secondary tumorsphere formation, 200 cells per well were plated in ultralow attachment 96-well plates. Cells were grown in StemXVivo Serum-Free Media (R&D) containing 2 U/ml Heparin (Tocris) and 0.8 μg/ml Hydrocortisone (Tocris). Mammospheres were harvested 7 days later, and tumorsphere was calculated under inverted phase-contrast light microscope (Olympus). The experiment was repeated three times. Statistical difference on secondary tumorsphere formation was assessed using student T-test with p ≤ 0.05 as the significance criterion.
When the effect of IL6 on tumorsphere formation was examined, recombinant human IL6 (Peprotech) was supplemented at concentration of 0, 100, 200 ng/μl in the beginning of the primary and secondary tumorsphere formation stages.
When the effect of IL6 on tumorsphere formation was examined, recombinant human IL6 (Peprotech) was supplemented at concentration of 0, 100, 200 ng/μl in the beginning of the primary and secondary tumorsphere formation stages.
5
Tumorsphere Formation and Characterization
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Caki-2 or A498 cells were seeded on ultralow attachment 6-well plates (Corning, NY, USA) at 5×103 cells per well for primary tumorsphere formation. After incubation for 2 weeks, tumorspheres were collected and enzymatically dissociated by trypsin. For secondary tumorsphere formation, 2000 cells per well were plated in ultralow attachment 96-well plates again. Cells were grown in StemXVivo Serum-Free Media (R&D systems) supplemented with 2 U/ml heparin (H8060, Solarbio, Beijing) and 0.8 μg/ml hydrocortisone (G8450, Solarbio, Beijing). Two weeks later, tumorspheres were observed and analyzed under an inverted phase-contrast light microscope (Olympus).
6
Tube Formation Assay for HUVECs
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Plates with 24 wells were first coated with Matrigel (BD Biosciences). Unpolymerized Matrigel was placed in the wells (300 μl/well) and allowed to polymerize for 1 h at room temperature. HUVECs in 500 μl medium were seeded onto the polymerized Matrigel at a density of 5 ×104 cells/well. After incubation with or without 10 ng/ml VEGF165, 1 mg/ml TA, 5 μg/ml VEGFR-1 and/or VEGFR-2 neutralizing antibody for 48 h, images of tube formation were acquired with an inverted phase- contrast light microscope (Olympus Corporation, Tokyo, Japan). The degree of tube formation was quantified in 5 random fields from each well using ImageJ software.
7
Histological Analysis of Scar Tissue
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Scar tissue samples were fixed in 4% paraformaldehyde for 24 h at 4°C and then dehydrated by washing with a series of ethanol solutions (50% ethanol for 2 h; 70% ethanol for 2 h; 80% ethanol for 2 h; 95% ethanol I for 2 h; 95% ethanol II for 1.5 h; 100% ethanol I for 1 h; 100% ethanol II for 30 min) at room temperature. Subsequently, samples were embedded in paraffin wax and cut into 5-µm sections for routine H&E and Masson staining. H&E staining was performed by staining with hematoxylin dye (10 min; room temperature) and eosin dye (1 min; room temperature) to observe the morphological changes of scar tissue. All slices were imaged using an inverted-phase contrast light microscope (Olympus Corporation; magnification, ×4 and ×20). Scar tissue fibrosis was detected by Masson staining using hematoxylin dye (10 min; room temperature), masson dye (10 min; room temperature) and 1% light green aqueous solution (5 min; room temperature). ImageJ software (version 1.52a; National Institutes of Health) was used to measure the epidermal thickness and Masson-positive area.
8
Angiogenesis Assay on Matrigel
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Plates with 24 wells were first coated with Matrigel (BD Biosciences). Unpolymerized Matrigel was placed in the wells (300 µL/well) and allowed to polymerize for 1 hour at room temperature. Human umbilical vein endothelial cells in 500 µL medium were seeded onto the polymerized Matrigel at a density of 5 × 104 cells/well.24 (link) After stimulated overnight, images of tube formation were acquired with an inverted phase-contrast light microscope (Olympus Corporation, Tokyo, Japan). The degree of tube formation was quantified in 5 random fields from each well at 40× magnification, using ImageJ software (version 1.48, National Institutes of Health, Bethesda, Maryland).22 (link)
9
Cellular Morphology Changes with Plasma Treatment
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4T1 and L929 cells were separately seeded in 6-well culture plates (2.5 × 105 cells/well) and incubated for 24 h. Then, the wells were washed three times with PBS (Sigma, USA). An inverted phase-contrast light microscope (Olympus, Japan) was used to capture photographs before treatment. Then, the wells were divided into three groups including control, hibernating plasma, and non-hibernating plasma. The untreated cells (0 mg/ml) were used as the control group, while the cells of hibernating plasma group were treated with the optimal hibernating plasma concentration selected from the MTT assay (16 mg/ml). The same concentration of non-hibernating plasma (16 mg/ml) was used to treat the cells of hibernating plasma group. At least, three wells were prepared for each group. After 24 h, the wells were washed three times with PBS and microscopic photographs were captured to evaluate changes in cellular morphology.
10
Colony Formation Assay Protocol
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Cell suspensions were plated in 60-mm culture dishes (300 cells per dish) and cultured for 2 weeks at 37°C. Subsequently, cells were fixed using 70% ethanol solution at room temperature for 15 min, after which, cells were stained using 0.005% crystal violet at room temperature for 30 min (Thermo Fisher Scientific, Inc.). The number of colonies with >50 cells was counted by an inverted phase contrast light microscope (magnification, ×200; Olympus Corporation).
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