A375 and B16-F10 CICs were isolated based on ALDH activity as previously described [37 (link)]. Gates for each sample were based upon N, N-diethylaminobenzaldehyde (DEAB) controls for each cell line. Sorted cells were cultured in low-adherent 6-well plates (Corning) in serum-free media at a density of 1 × 103cells/mL. Cultures were grown for up to 14 days and treated with fresh media containing either 100 μM Lunasin or vehicle (PB) twice per week. Oncospheres (> 100 μm) were counted and imaged using an EVOS light microscope (Life Technologies) and images were analyzed using Image-J software (NIH) as described [37 (link)].
Evos light microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EVOS light microscope is a compact and versatile instrument designed for a wide range of imaging applications. It features LED illumination and high-resolution optics to provide clear, detailed images of samples. The EVOS is capable of brightfield, phase contrast, and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Low adherent 6 well plates, by Corning (2 mentions)
Paraffin, by Electron Microscopy Sciences (1 mentions)
M205 stereoscope, by Leica (1 mentions)
Permount mounting medium, by Thermo Fisher Scientific (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Poly hema, by Merck Group (1 mentions)
Methocult classic, by STEMCELL (1 mentions)
7 protocols using evos light microscope
1
Isolation and Culture of Cancer Stem Cells
2
Lung Metastasis Histological Analysis
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After fixation in 10% formalin, lungs were transferred to 70% ethanol and stored overnight at room temperature. Tissues were dehydrated through a series of graded alcohols, and infiltrated with paraffin (Electron Microscopy Sciences). Tissues were embedded in paraffin and sectioned (thickness = 7 μm) on a microtome. Sections were transferred to SuperFrost Plus slides (Fisher) and allowed to dry overnight on a slide warmer (Fisher). paraffin removal was initiated by several washed in xylene, and followed by rehydration of the tissues in a series of graded alcohols. Tissues were stained in hematoxylin and eosin (H&E) solutions followed by a clearing solution of xylene. After staining, PermountTM mounting medium (Fisher) was applied to each slide and covered with a 60 mm cover slip (Fisher). Slides were allowed to dry at room temperature overnight and then placed in a drying oven until completely dry. Images of H&E stained slides were taken using a Leica M205 Stereoscope (Leica) as well as an EVOS light microscope (Life Technologies). Macrometastases were counted under 4.32x magnification on the Leica M205 Stereoscope. Micrometastases were counted from H&E stained non-sequential sections (n = 5) from each tissue sample using an EVOS light microscope. Images were subsequently analyzed for total tumor area using ImageJ software (NIH).
3
Culturing B16-F10 Oncospheres
4
Oocyte Diameter Measurement and Analysis
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The diameter of oocytes was measured before storage using the image report system of the EVOS light microscope (Life technologies) and the imaging software ImageJ. Differences in oocyte diameter were determined by non-parametric Kruskall-Wallis and Dunn’s multiple comparisons tests using GraphPad Prism version 5.0. Differences were considered significant when p < 0.05.
5
Quantifying αSyn Pathology in CNS
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Paraffin-embedded brain sections were stained with anti GFAP and anti Iba-1 antibodies, images captured by Aperio XT scanner and analyzed using the Positive Pixel Count algorithm in ImageScope (Aperio). At least three slides were used to calculate an averaged immunostaining burden quantity by a blinded experimenter.
CNS distribution of αSyn pathology was performed by a blinded experimenter by visually evaluating slides from each experiment (4 month or 2 month cohorts) in a single sitting on an Evos light microscope (ThermoFisher). A secondary reviewer also performed random spot-checks to establish colorimetric assessment. Presence of αSyn pathology is represented by red dots on a brain map to reflect the relative levels of pathology in a qualitative manner. To quantitatively assess absolute aggregate counts, the number of EP1536Y and p62 immunoreactive inclusions were counted from different samples corresponding to slides that were all cut at a given neuroanatomic location.
CNS distribution of αSyn pathology was performed by a blinded experimenter by visually evaluating slides from each experiment (4 month or 2 month cohorts) in a single sitting on an Evos light microscope (ThermoFisher). A secondary reviewer also performed random spot-checks to establish colorimetric assessment. Presence of αSyn pathology is represented by red dots on a brain map to reflect the relative levels of pathology in a qualitative manner. To quantitatively assess absolute aggregate counts, the number of EP1536Y and p62 immunoreactive inclusions were counted from different samples corresponding to slides that were all cut at a given neuroanatomic location.
6
Spheroid Formation in Ovarian Cancer
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ES-2, ES-2-Rv-Ctrl, ES-2-Rv-NICD3 cells and a population of ES-2 cells combined with ES-2-Rv-NICD3 (ES-2:ES-2-Rv-NICD3, 1:3) were plated at 10,000 cells/well on 24 well plates coated with polyHema (30 mg/mL, Sigma-Aldrich) with or without 1000 kDa HA (50 µg/mL, Contipro Inc., Dolní Dobrouč, Czechia). After 72 h, spheroids were imaged by light microscopy (Olympus IX-71, 5 images/well) and spheroid area was quantitated using FIJI (FIJI 2, version 2, Cambridge Astronomical Survey Unit). OV90 and OVCAR3 cells were plated at 50,000 cells/well with or without 1000 kDa HA (50 µg/mL) for 72 h before imaging by EVOS light microscope (Thermo Fisher Scientific, Waltham, MA, USA), 5 images/well. A2780 and OVCAR3 (Lv-Ctrl or Lv-DAB2, 50,000 cells/well) were cultured 3 and 5 days respectively before imaging.
7
Dual Editing of CD34+ Cells for Clonogenic Assay
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CD34+ cells which had undergone DNAm targeting alone or dual genetic and epigenetic editing were cultured in StemSpan II supplemented with cytokines for 24 h before seeding into the CFU assay. CD34+ cells were counted, and 600 cells were seeded per plate into MethoCult Classic (StemCell Technologies) after DNAm editing, or 1,200 cells per plate after dual editing. Colonies were grown in an incubator for 14 d before counting and harvesting either bulk or single colonies. For single-colony harvest, individual colonies were picked using an EVOS light microscope (ThermoFisher Scientific) and deposited directly into a PCR tube for analysis.
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