Cells were seeded at a density of 1 x 105 cells/250 µL complete MEMα into each well of a 24-well plate and incubated for 24 h. The complete MEMα was then replaced with 500 µL fresh complete MEMα and the plate was incubated for 48 h. The media was replaced with vehicle or BMM and incubated for 72 h. This process was continued every other day for a total of 14 days from first treatment. Half of the wells were harvested for mRNA. The remaining wells were stained with Alizarin Red S (ARS) to quantify total mineralization. Briefly, cells were fixed with 10% neutral buffered formalin for 5 min at room temperature and then stained with 2% ARS (Sigma-Aldrich, Cat #: A5533-25G) at pH 4.1 in water (2 g/100 mL) for 30 min at room temperature. Excess stain was washed out in running tap water until the water ran clear. Representative images were captured on an EVOS inverted microscope (Thermo Fisher Scientific). Stain from each wells was quantified after lysing entire contents as described elsewhere using an acetic acid extraction method and plate reader absorbance at 405 nm [16] (link).
Evos inverted microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EVOS inverted microscope is a high-performance imaging system designed for cell culture and live-cell imaging applications. It features a motorized stage, various contrast enhancement methods, and an intuitive software interface for seamless image capture and analysis.
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Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Apotome, by Zeiss (2 mentions)
Tcs sp5, by Leica (1 mentions)
Incucyte zoom, by Sartorius (1 mentions)
Click it plus edu imaging kit, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
96 well tissue culture plate, by Corning (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Polycarbonate membrane, by Neuro Probe (1 mentions)
Fetal bovine serum (fbs), by Miltenyi Biotec (1 mentions)
Dulbecco modified eagle medium (dmem), by Miltenyi Biotec (1 mentions)
Methanol, by Avantor (1 mentions)
Crystal violet, by Thermo Fisher Scientific (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Axio observer z1 with apotome, by Zeiss (1 mentions)
Mouse rankl, by R&D Systems (1 mentions)
Transwell boyden chamber, by Corning (1 mentions)
18 protocols using evos inverted microscope
1
Osteogenic Differentiation Assay
2
Wound Healing Assay with OVCAR Cell Lines
3
Scratch Assay for Cell Migration
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In 12-well plates,
2 × 105 cells per well were grown in monolayers to
full confluence. A longitudinal scratch was made with a sterile pipette
tip at the bottom of each well. Wells were washed with PBS and cells
were treated with the corresponding medium with mA4 at concentrations
of 2.5 and 5 μM and without mA4 (control). As an assay control,
cellular migration was also evaluated after incubation of A4 at a
concentration of 2 μM. Fetal bovine serum was not used for the
treatments, as it could degrade the aptamers.
Images were acquired
using an EVOS inverted microscope (Thermo Fisher Scientific, Waltham,
MA, USA) at 0, 24, and 48 h after the treatments. The open (wounded)
areas of the images were calculated using the ImageJ program (v1.52o,
NIH, Bethesda, MD, USA) to assess cell migration.
2 × 105 cells per well were grown in monolayers to
full confluence. A longitudinal scratch was made with a sterile pipette
tip at the bottom of each well. Wells were washed with PBS and cells
were treated with the corresponding medium with mA4 at concentrations
of 2.5 and 5 μM and without mA4 (control). As an assay control,
cellular migration was also evaluated after incubation of A4 at a
concentration of 2 μM. Fetal bovine serum was not used for the
treatments, as it could degrade the aptamers.
Images were acquired
using an EVOS inverted microscope (Thermo Fisher Scientific, Waltham,
MA, USA) at 0, 24, and 48 h after the treatments. The open (wounded)
areas of the images were calculated using the ImageJ program (v1.52o,
NIH, Bethesda, MD, USA) to assess cell migration.
4
Confocal Microscopy for Subcellular Localization
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Cells were imaged with the confocal microscope Leica TCS SP5 fitted with the HCX APO 63×/1.30 Corr (glycerol) CS 21objective or Evos inverted microscope (Thermo Fisher Scientific). Where applicable, experiments were run in at least duplicates and repeated at least three times with similar results. Micrographs were collected at least 10 frames per sample. The data set was blinded and analyzed. The data are represented as normalized between experiments or single experiments where repetitions show similar results.
Images were analyzed using Fiji (NIH). For co-localization studies, confocal images were automatically analyzed and individual cells were manually selected as region of interest. The data were analyzed using JASP software. The data are shown as mean values with standard deviation as error bars. Statistical significance was investigated by non-paired Student’s t-test or one-way ANOVA, followed by Bonferroni’s post hoc test (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001).
Images were analyzed using Fiji (NIH). For co-localization studies, confocal images were automatically analyzed and individual cells were manually selected as region of interest. The data were analyzed using JASP software. The data are shown as mean values with standard deviation as error bars. Statistical significance was investigated by non-paired Student’s t-test or one-way ANOVA, followed by Bonferroni’s post hoc test (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001).
5
Wound Healing and 3D Invasion Assays
6
Scratch Assay for Cell Migration
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The OVCAR5 NTC and sh1 KD cells and OVCAR7 EV and LRRC15 OE cells were scratched with 10 μL pipette tips and replaced with fresh media. Zero-hour/24-hour images were captured using an EVOS inverted microscope (Thermo Fischer Scientific).
7
Wound Healing Assay with Transduced Cells
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For the wound healing assay, EPC2 cells and HaCaT cells transduced with DSP or PPL constructs were simultaneously seeded on a Culture-Insert 4 Well in μ-Dish 35 mm, high ibiTreat (Ibidi USA), grown until confluency. The Culture-Insert 4 Well allows for wound healing assays with four cell-free gaps with a 0.5 mm gap width and one center area with a 1 mm diagonal distance. After removal of the insert, cells were washed three times, incubated at 37 °C and 5% CO2, and observed at 8 h or 12 h post wounding. Each wound was imaged at the time of wounding (0 h) and 8 h or 12 h post wounding using an EVOS inverted microscope (Thermo Fisher Scientific). The gap area was quantified using the ImageJ plugin MRI Wound Healing Tool (http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool ). Percent change in wound area was defined as [100% × (W0-W8)/W0 (W0: wound width at 0 h; W8: wound width at 8 h)].
8
Comprehensive Cellular Assays for Stemness
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For clonogenicity assays, cells were plated in triplicate onto 60‐mm dishes at low density (1,000 cells/dish) and cultured for 8–10 days. Colonies were fixed with 4% formaldehyde and stained with 0.1% crystal violet. The number and size of colonies were counted by Fiji software.
For sphere‐forming assays, cells were plated onto 8‐well chamber slides (1,000 cells/well) pre‐coated with 80 µl of Matrigel (BD Biosciences) for 1 h at 37°C. After 8 days, slides were fixed with 4% formaldehyde for 20 min at RT and images were acquired using EVOS inverted microscope (Thermo Fisher Scientific). Number and size of spheres were counted by Fiji software.
For live‐cell proliferation assays, cells were plated in triplicate at low density (1,000 cells/well) into 96‐well trays. Plates were placed into IncuCyte ZOOM (Sartorius) for 5 days, with pictures and cell density measurements taken every 2–3 h.
5‐Ethynyl‐2′‐deoxyuridine (EdU) incorporation assays were performed using Click‐iT Plus EdU Imaging Kit (Thermo Fisher Scientific) following the manufacturer’s instructions, with nuclear staining by DAPI (Sigma, D9542, 1:1,000 dilution in PBS, 10 min incubation). Fraction of EdU‐positive cells was determined by Fiji software.
For sphere‐forming assays, cells were plated onto 8‐well chamber slides (1,000 cells/well) pre‐coated with 80 µl of Matrigel (BD Biosciences) for 1 h at 37°C. After 8 days, slides were fixed with 4% formaldehyde for 20 min at RT and images were acquired using EVOS inverted microscope (Thermo Fisher Scientific). Number and size of spheres were counted by Fiji software.
For live‐cell proliferation assays, cells were plated in triplicate at low density (1,000 cells/well) into 96‐well trays. Plates were placed into IncuCyte ZOOM (Sartorius) for 5 days, with pictures and cell density measurements taken every 2–3 h.
5‐Ethynyl‐2′‐deoxyuridine (EdU) incorporation assays were performed using Click‐iT Plus EdU Imaging Kit (Thermo Fisher Scientific) following the manufacturer’s instructions, with nuclear staining by DAPI (Sigma, D9542, 1:1,000 dilution in PBS, 10 min incubation). Fraction of EdU‐positive cells was determined by Fiji software.
9
Mouse Prostate Organoid Isolation and Growth
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Mouse prostate tissue isolation and organoid growth was carried out as described by Drost et al. [25] , with selection of EpCAM positive epithelial cells, as described in detail in the supplementary information. Brightfield and fluorescent organoid images were taken with an EVOS inverted microscope (ThermoFisher Scientific, UK).
10
Cytotoxicity Evaluation of ML-II in Lung Cancer Cells
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The MTS assay was performed to determine the viability of the A549 and H460 cells treated with ML-II. The cells were incubated in 96-well tissue culture plates (Corning, NY, USA) at a density of 10 × 103 cells/well in 200 µl at 37 °C in a humidified, 5% CO2 atmosphere incubator. After 24 h of incubation, the cells were treated with ML-II or vehicle (PBS) starting at a concentration of 50 µg/mL. The cytotoxic effects were measured after 96 h of incubation. The media were removed and replaced with PBS supplemented with 4.5 g/L glucose, which was followed by the addition of 20 µL of the MTS solution to every well and incubation for 1–4 h at 37 °C [43 (link)]. The production of formazan by live cells was determined by measuring the OD at 490 nm. Cell viability was calculated as follows: % viability = absorbance of treated/absorbance of untreated × 100. In addition, morphological changes were assessed under an EVOS inverted microscope (Thermo Fisher, Waltham, MA, USA).
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