Morphological changes after 48 h of cancer cells (2x106 cells/ml) treated with sample (25 μg/ml) along with the untreated control were observed under an inverted microscope (Labomed, Germany) (Freshney 2000 ).
Inverted microscope
Manufactured by Labomed
Sourced in United States
The Inverted microscope is a type of optical microscope that has the objective lens positioned below the specimen stage. This design allows for the observation of cells or tissues in a culture dish or other container from the underside. The inverted microscope is a versatile instrument commonly used in cell biology, tissue culture, and other applications where a top-down view of a sample is preferred.
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Lab products found in correlation
Absorbance microplate reader, by Agilent Technologies (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
24 well plate, by SPL Life Sciences (1 mentions)
Hisep media, by HiMedia (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Cell culture flask, by SPL Life Sciences (1 mentions)
Dmem medium, by Biosera (1 mentions)
Antibiotic antimycotic solution, by Biosera (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
13 protocols using inverted microscope
1
Morphological Changes in Cancer Cells
2
Wound Healing Assay with Sorafenib, Avastin, and Fucoidan
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Briefly, 106 HUH-7 cells were seeded in six 6-well plates and allowed to attach overnight (o/n). Once cells reached confluency, the cell monolayer was scratched using a 200 μL pipette tip held vertically followed by washing twice with PBS to remove floating cells. The cells in each plate were then treated with complete DMEM medium alone (untreated control) or added to it either 5 µM sorafenib, 25.22 µM Avastin, 55 μg/ml fucoidan or the combinations of sorafenib and fucoidan (S + F) or Avastin and fucoidan (A + F). The wound area was imaged on day 0, 1, 2, 3 and 4 post treatment using an inverted microscope (Labomed Inc., LA, CA, United States) connected to a digital camera. Wound width was calculated using a wound healing size plugin for ImageJ® (NIH, United States) as described by (Suarez-Arnedo et al., 2020 (link)).
3
Alizarin Red Staining for Calcium Deposition
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Alizarin red staining was accomplished to detect calcium deposition in the extracellular matrix after 21 days. Because of the presence of calcium in scaffolds, it was difficult to distinguish calcium deposition in ECM. Thus, BMSCs were cultured in the plate, and conditioned media (10% FBS-supplemented DMEM containing 0.1 mg/mL of each scaffold) were added. After 21 days, media was removed; the BMSCs were washed with PBS, and fixed with 4% paraformaldehyde (PFA) for 40 min at room temperature. Then, PFA was pulled out and replaced with alizarin red solution (2% (w/v) in deionized water, pH 4.1–4.3). After incubation at 4 °C for 40 min, the stained BMSCs were observed under an inverted microscope (Labomed, USA).
4
Ovarian Follicle Development with CoQ10 Supplement
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In both groups the ovaries were cultured on the insert (0.4-μm pore size; Greiner Bio-One, Germany) in the 24-well plates (SPL, Korea) containing 300 μl of α-MEM supplemented with 10% FBS, 1% insulin, transferrin and selenium (ITS; Gibco, USA), 100 mIU/mL recombinant FSH (rFSH; Homa pharmed; Iran) in the CO2 incubator (5% CO2, 100% humidity, 37°C) for one weethe k. In experimental group 50 μM CoQ10 (Nono kimia; Kore) was added to the culture media (12 (link), 14 ). Every other day, the development of ovarian follicles was evaluated under an inverted microscope (Labomed, USA) and, half of the culture media (150 μl) was renewed. The collected culture medium was stored at −20°C until measurement of the 17-β-estradiol level.
5
Wound Healing Assay with Drugs
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Briefly, 106 HepG2 cells were seeded in 6-well plates and allowed to attach overnight. Once the cells reached confluency, a wound was made by scratching the surface with a 200 μL pipette tip held vertically. To remove floating cells, the cells were washed twice with PBS. The cells were then treated with complete DMEM medium and either 3 mM MET, 344.45 μM TMP or 0.2 mM MTX or the combinations MET + TMP and MET + MTX (lower concentrations were used to avoid the detachment of cells). The initial wound area was measured at time 0 using an inverted microscope (magnification power of 400x) (Labomed Inc., LA, CA, USA) connected to a digital camera. The wound distance was then assessed by ImageJ software.
6
Three-Day MDA-MB-231 Spheroid Formation
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MDA-MB-231 cells harvested and single-cell suspensions at a density of 1 × 104 cells per 20 μl of RPMI culture medium containing 1% FBS, 0.1% gelatin carefully placed under the lids of cell culture plate and were inverted over the culture plates. Then the plates were incubated at 37 °C under a humidified condition with 5% CO2. After 3 days of incubation, the spheroids formation was monitored by using the inverted microscope (Labomed, USA).
7
Quercetin's Cytotoxic Effects on HeLa and Lymphocytes
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Approximately 10000 HeLa cells/well were plated in 96-well plate and incubated for 24 h. After attachment, the cells were treated with different concentrations of quercetin ranging from 1 to 150 µM for 24 and 48 h. Similarly, cells were treated with vehicle control using DMSO. Morphological changes in HeLa cells were recorded using an inverted microscope (Labomed, U.S.A.). Following the treatment, MTT (Sigma–Aldrich) at final concentration of 0.5 mg/ml was added and incubated at 37°C for 2 h. The formazan crystals were solubilized with 100 µl of DMSO with 20-min incubation at 37°C (Sigma–Aldrich). Absorbance Microplate Reader (BioTek, U.S.A) was used to record the absorbance at 570 nm and calculate the viability of the cells. The experiments were repeated thrice and expressed as an average. The cell viability was calculated following the below-mentioned formula:
Lymphocytes were isolated from fresh blood using HiSep Media (HiMedia, India) following the manufacturer’s instructions. They were then resuspended in RPMI media and plated in 96-well microplates at approximately 10,000 cells/well and treated with quercetin as stated above. MTT assay was performed after 24 h exposure.
Lymphocytes were isolated from fresh blood using HiSep Media (HiMedia, India) following the manufacturer’s instructions. They were then resuspended in RPMI media and plated in 96-well microplates at approximately 10,000 cells/well and treated with quercetin as stated above. MTT assay was performed after 24 h exposure.
8
Collection and Maturation of Cumulus Oocyte Complexes
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The collection and in vitro maturation of cumulus oocyte complexes (COCs) were done according to Totey et al21 with some modifications. Briefly, COCs were aspirated from visible large antral follicles having diameter of 4‐6 mm using a syringe (10 mL; Henke Sass Wolf) and needle (18 G; Henke Sass Wolf). COCs with oocyte of 120‐125 µm in diameter (excluding the zona pellucida) were selected with the help of a stereomicroscope. Diameters of oocytes were measured using an ocular micrometer attached to an inverted microscope (Labomed, Inc). COCs containing healthy oocytes were selected based on their morphological appearance (uniformly granulated cytoplasm surrounded by multilayered compact cumulus cells) for further experimentation.
9
Tumorsphere Formation Assay
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Cells (3 × 104) from single cell suspensions were cultured in a serum free medium containing DMEM/F12 (Gibco, UK) supplemented with 10 µM basic fibroblast growth factor (bFGF, Sigma-Aldrich, USA) and 10 µM epidermal growth factor (EGF, Sigma-Aldrich, USA) in non-treated 6 well culture plates (SPL, Korea). Culture media were replaced or supplemented with fresh growth factors every three days. After 7 days, each well was examined for formation of tumorsphere-like cell aggregates by an inverted microscope (Labomed, USA).
10
Isolation and Characterization of Human Dermal Fibroblasts
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Human dermal fibroblast cells were purchased from Bon Yakhte Corp. (Tehran, Iran). First, the cells were examined for CD34, CD45, CD73, CD90 and CD105 biomarkers expressions by flowcytometry. 4 mL of high glucose content (4.5 g/L) DMEM medium (Biosera, France) containing 10% FBS (Gibco, USA) and 1% antibiotic-antimycotic solution (Biosera, France) were used for cell culture. The culture medium in the Falcon was filled with the cell suspension, which was then carefully pipetted into the container. The cell-containing tube was then spun at 230 g for 5 minutes (VISION SCIENTIFIC, South Korea). After removing the supernatant, the cell sediment was suspended in 1 mL of culture medium, and transferred to a 25 cm cell culture flask (SPL, South Korea) containing 4 mL of complete culture medium. After observing the cells inside the flask under an inverted microscope (LaboMed, USA), the flask was transferred to an incubator (MEMMERT, Italy) with a temperature of 37 °C, 5% CO2, and 95% relative humidity (RH).
The percentage of viable cells was determined by cell staining with trypan blue. For this purpose, at first 1 mL cell suspension was prepared, and 20 µL of trypan blue 0.25% was added to cell suspension and poured into a well of a 96-well plate. About 10 µL of the mixture was placed on Neubauer slide and transferred under the microscope.
The percentage of viable cells was determined by cell staining with trypan blue. For this purpose, at first 1 mL cell suspension was prepared, and 20 µL of trypan blue 0.25% was added to cell suspension and poured into a well of a 96-well plate. About 10 µL of the mixture was placed on Neubauer slide and transferred under the microscope.
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