Briefly, placenta sections were immunostained with primary H3K27me3 (1:500) antibodies at 4°C overnight and HRP‐labelled goat anti‐rabbit IgG the next day. Staining was completed by incubation with diaminobenzidine chromogen solution (DAB; Solarbio). The sections were counterstained with Harris's haematoxylin (Solarbio), and then dehydrated and mounted. Images were captured on an inverted phase microscope (Leica Microsystems GmbH).
Inverted phase microscope
Manufactured by Leica
Sourced in Japan
The Inverted Phase Microscope is a type of optical microscope that uses phase contrast illumination to enhance the visibility of transparent or colorless specimens. It is designed with the light source and condenser positioned below the stage, allowing for the observation of samples from below. This configuration enables the examination of living cells or other specimens in culture dishes or petri dishes.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Heparin, by Roche (1 mentions)
Recombinant human egf, by Merck Group (1 mentions)
Recombinant human bfgf, by Merck Group (1 mentions)
Transwell chamber, by Corning (1 mentions)
Inverted microscope, by Leica (1 mentions)
Low melting temperature agarose, by Lonza (1 mentions)
6 protocols using inverted phase microscope
1
Immunohistochemical Analysis of H3K27me3
2
Neurosphere Formation Assay for Cancer Stem Cells
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Neurosphere formation was used as an established experimental assay to evaluate cancer stem cell proliferation (Nör et al. 2013; Zanini et al. 2013) . Daoy and D283 cells were dissociated with trypsin-EDTA into a single cell suspension and seeded at 500 cells/well in 24-well low-attachment . CC-BY-NC 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not this version posted January 30, 2020. ; https://doi.org/10.1101/521393 doi: bioRxiv preprint 10 plates. The cells were cultured in serum-free sphere-induction medium, containing DMEM/F12 supplemented with 20 ng/ml epidermal growth factor (EGF, Sigma-Aldrich), 20 ng/ml basic fibroblast growth factor (Sigma-Aldrich), B-27 supplement 1X (Gibco), N-2 supplement 0.5X (Gibco), 50 μg/ml bovine serum albumin (Sigma Aldrich), and antibiotics during 7 days as previously described (Nör et al. 2013 ). The ERK inhibitor U0126 was added in the first day after plating cells with the sphere-induction medium. Spheres photomicrographs were captured 7 days after treatment under an inverted phase microscope (Leica Microsystems, Mannheim Germany) at ×10 magnification.
The copyright holder for this preprint (which was not this version posted January 30, 2020. ; https://doi.org/10.1101/521393 doi: bioRxiv preprint 10 plates. The cells were cultured in serum-free sphere-induction medium, containing DMEM/F12 supplemented with 20 ng/ml epidermal growth factor (EGF, Sigma-Aldrich), 20 ng/ml basic fibroblast growth factor (Sigma-Aldrich), B-27 supplement 1X (Gibco), N-2 supplement 0.5X (Gibco), 50 μg/ml bovine serum albumin (Sigma Aldrich), and antibiotics during 7 days as previously described (Nör et al. 2013 ). The ERK inhibitor U0126 was added in the first day after plating cells with the sphere-induction medium. Spheres photomicrographs were captured 7 days after treatment under an inverted phase microscope (Leica Microsystems, Mannheim Germany) at ×10 magnification.
3
Tumorsphere Expansion Assay with RA Induction
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A tumorsphere expansion assay was performed as previously described [24 (link)]. Briefly, cells were plated at 1000 cells/well in 24-well plates using DMEM/F12 (1:1) sphere induction medium containing 2% of B27 (Gibco), 20 ng/mL recombinant human bFGF (Sigma-Aldrich), 20 ng/mL recombinant human EGF (Sigma-Aldrich), heparin 10 IE/mL (5 mg/mL) (Roche, Mannheim, Germany), and antibiotics. After three days of sphere formation, RA was added at 10 μM concentration. Sphere photomicrographs were captured at day seven under an inverted phase microscope (Leica Microsystems, Mannheim, Germany) at ×5 magnification. Spheres were measured using ImageJ. The spheres’ RNAs were also collected for RT-qPCR analysis.
4
Transwell Invasion Assay for H1299 Cells
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Transwell assay was carried out using transwell chambers (Corning) for detecting the invasiveness of H1299 cells and performed using 24 well transwell plates with polycarbonate membrane and 4.0 μM pores (Corning). After 24 h of LSD1 inhibitors treatment or 48 h of transfection, H1299 cells in serum-free RPMI 1640 medium were seeded in chambers with solidified matrigel (4.0 μg/μL, 60 μL) in advance for 3 h at RT. RPMI 1640 medium with 10% FBS was added to the lower chambers. H1299 cells in the upper chambers were at a density of 500 cells/per well. After 24 h, the medium from the lower chamber was removed and the upper chamber cells were wiped, then the migrated cells were fixed in 4% paraformaldehyde and stained with crystal violent for 30 min at RT. Migrating cell numbers were observed in four random fields under an inverted phase microscope (Leica) and cell numbers were enumerated using Image J.
5
Evaluating EV-Driven Cell Migration
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For evaluation of the ability of EVs to promote cell migration, HUVECs were seeded in 6-well culture plates. After the cells reached more than 85% confluence, the monolayer of HUVECs was scraped using a 200 μL micropipette tip. Then, 3 mL of ECM containing different types of EVs was added to each well, and the cells were incubated for an additional 24 h. Cell images at 0 h and 24 h were taken using an inverted phase microscope (Leica, Japan). The migration rate (%) of cells was calculated as follows: Migration rate (%) = (A0 – An)/A0 × 100%. A0 represents the initial wound area, and An represents the final wound area.
Transwell (8.0 μm pore size, Corning Company) tests were used to assess the number of cells that migrated. HUVECs or EPCs in 200 µL of serum-free medium were seeded into the upper chamber. Complete medium (400 μL) with different types of EVs or PBS was added to the lower chamber. After 12 h, pictures of the five regions were taken with an inverted microscope (Leica, Japan). The number of migrated cells was measured by ImageJ (Version 1.5.3).
Transwell (8.0 μm pore size, Corning Company) tests were used to assess the number of cells that migrated. HUVECs or EPCs in 200 µL of serum-free medium were seeded into the upper chamber. Complete medium (400 μL) with different types of EVs or PBS was added to the lower chamber. After 12 h, pictures of the five regions were taken with an inverted microscope (Leica, Japan). The number of migrated cells was measured by ImageJ (Version 1.5.3).
6
Soft Agar Colony Formation Assay
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Soft agar assays were carried out in a 6‐well plate, each sample in triplicate and colonies were counted on day 15 after plating. The base layer consisted of 2 mL with a final concentration of culture medium and 0.6% low melting temperature agarose (Lonza). Next, the respective cells were seeded with culture medium containing 0.6% agar to result in a final concentration of was 0.3% agarose with 5 x 104 cells. A further 500 μL culture medium only was added above the congealed middle agar layer on the next day. The colonies were captured at five random fields at 40× magnification with inverted phase microscope (Leica), measured using the Leica software. The scale was set at ImageJ to select colony size more than 0.45 mm.
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