Osteosarcoma cells (U2OS, obtained from ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and antibiotics. GM06865 lymphocytes (obtained from Coriell Biorepository) were maintained in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 15% FBS and antibiotics. Both types of cells were cultured at 37°C in an atmosphere of 5% CO2. For replication stress analysis, asynchronously growing cells were seeded onto glass coverslips (Sigma-Aldrich) and were treated with 0.2 mM hydroxyurea (HU; Sigma-Aldrich) for 16 hours. For karyotype analysis, asynchronously growing cells were treated with 0.1 µg/ml colcemid (Karyomax, Thermo Fisher Scientific) for 5 hours before being harvested.
Glass coverslips
Manufactured by Merck Group
Sourced in United States
Glass coverslips are thin, transparent plates made of glass. They are used to cover and protect samples or specimens for microscopic observation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Gold nanoparticle solution, by Merck Group (2 mentions)
Stir bars, by Merck Group (2 mentions)
Uric acid, by Merck Group (2 mentions)
20 ml glass vial, by Merck Group (2 mentions)
Poly methyl methacrylate pmma discs, by McMaster-Carr (2 mentions)
Direct q3 uv apparatus, by Merck Group (2 mentions)
L alanine, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Karyomax, by Thermo Fisher Scientific (1 mentions)
Anti rabbit fitc secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
13 protocols using glass coverslips
1
Characterizing Osteosarcoma and Lymphocyte Cells
2
Cellular Actin and EGFR Localization
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Phalloidin staining was performed to study actin, and EGFR was stained to study its cellular localization. Briefly, 2 × 105 cells were seeded in a six-well plate on slats and permeabilized with 4% paraformaldehyde, then blocked with a 10% PBS–foetal bovine serum solution. For phalloidin staining, cells were incubated in a 1% PBS–BSA solution with 1/5000 DAPI (Sigma-Aldrich, Saint-Louis, MO, USA) and 1/200 phalloidin-fluorescein antibody (Sigma-Aldrich). For EGFR staining, cells were incubated in 0.1% PBS–Triton solution for 2 h with 1/100 EGFR primary antibody (sc-03; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then cells were incubated in a 0.1% PBS–Triton solution for one hour with 1/200 anti-rabbit FITC secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 1/5000 DAPI. Sections were mounted using glass coverslips and mounting medium (Sigma-Aldrich), and then visualized on a Zeiss fluorescence microscope (AxioImager Z2, Zeiss, Oberkochen, Germany).
3
Rapid Microwave-Assisted Biosensing Assay
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l -Alanine, uric acid, gold nanoparticle solution (diameter = 20 nm, optical density = 1), glass coverslips, 20 mL glass vials, stir bars and thermometers were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was obtained from Millipore Direct Q3 UV apparatus (Billerica, MA, USA). Adhesive silicone isolators with 21-well capacity (depth = 2.0 mm, diameter = 4.5 mm with a capacity of 30 µL, and diameter = 3.5 mm with a capacity of 10 µL) were designed by the Aslan Research Group and manufactured by Grace BioLabs (Bend, OR, USA). Poly(methyl)methacrylate (PMMA) discs (5 cm diameter) were purchased from McMaster-Carr (Chicago, IL, USA). The iCrystal plates were created by combining the PMMA discs and the adhesive silicone isolators. Adhesive silicon isolator was carefully attached to the PMMA platform and gently pressed to ensure water-tight attachment. The 8 GHz medical microwave (maximum wattage 20 W) was obtained from Emblation Microwave (Inglewood, Alloa, Scotland, UK).
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4
Cellular Uptake Quantification of ICG
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LA-4 were seeded on glass coverslips (Sigma-Aldrich) positioned inside 12 multiwell plates. After treatment, cells were fixed with paraformaldehyde 4% and stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, Thermo Fisher Scientific) for 5 min. After three washings with PBS, the cytoplasmatic or nuclear ICG uptake (red) was evidenced with fluorescence microscope Nikon Eclipse Ti2 (Nikon) and cell nuclei were highlighted in blue.
5
Fluorescence Imaging of Receptor Signaling
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Isoproterenol , (±)‐propranolol, poly‐L‐lysine, glass coverslips, paraformaldehyde (PFA), and anti‐FLAG (RRID:AB_10950495) antibodies were purchased from Sigma‐Aldrich (Diegem, Belgium). Primary anti‐hemagglutinin antibodies (RRID:AB_390918) were purchased from Roche (Basel, Switzerland). d ‐Luciferin was purchased from Promega, UK. Secondary antibodies (donkey anti‐mouse coupled to AlexaFluor 488, fluorophore (RRID:AB_2556746) and goat anti‐rat antibodies coupled to AlexaFluor 555 fluorophore (RRID:AB_2535855)), together with bovine serum albumin, l ‐glutamine, penicillin/streptomycin, ammonium chloride, trypsin‐EDTA, and microscope slides were purchased from Thermo Fisher Scientific (Waltham, MA). Cell culture medium (Dulbecco's Modified Eagle Medium) and Puromycin (50 mg/mL Stock) were purchased from Invitrogen (Merelbeke, Belgium). 96‐well plates were purchased form Greiner Bio‐One (Wemmel, Belgium). 4′,6‐diamidino‐2‐phenylindole‐containing mounting medium was purchased from Biotium (San Francisco, CA). Fetal bovine serum (FBS) was purchased form Biowest (Riverside, MO).
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6
Isolation and Culturing of Rat Hippocampal Neurons
7
Live-Dead Cell Viability Assay for NSPCs
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A live-dead cell assay kit (Life Technologies) was used to assess NSPC viability,
according to previously established protocols6 . Briefly, cells were plated on poly-D-lysine/laminin (Sigma-Aldrich, St. Louis, MO,
USA)-coated glass coverslips (Sigma-Aldrich), and placed in 24 well plates with growth
medium. Media was subsequently removed and the cells exposed to 4 μM ethidium homodimer 1
and 2 μM calcein AM dye in 1× phosphate buffered saline (PBS; Life Technologies). After 45
min, the number of green cells (live, labeled with calcein AM dye) and red cells (dead,
labeled with ethidium homodimer 1) were counted in five random fields per coverslip under
a 20× lens. At minimum, n = 3 independent NSPC cultures, grown in
parallel, were assessed in triplicate for each age group of cells examined.
according to previously established protocols6 . Briefly, cells were plated on poly-D-lysine/laminin (Sigma-Aldrich, St. Louis, MO,
USA)-coated glass coverslips (Sigma-Aldrich), and placed in 24 well plates with growth
medium. Media was subsequently removed and the cells exposed to 4 μM ethidium homodimer 1
and 2 μM calcein AM dye in 1× phosphate buffered saline (PBS; Life Technologies). After 45
min, the number of green cells (live, labeled with calcein AM dye) and red cells (dead,
labeled with ethidium homodimer 1) were counted in five random fields per coverslip under
a 20× lens. At minimum, n = 3 independent NSPC cultures, grown in
parallel, were assessed in triplicate for each age group of cells examined.
8
Culturing A549 Lung Cancer Cells
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The human lung cancer cell line A549 were routinely propagated as follows: DMEM medium, with 10% fetal calf serum (FCS), 2% glutamine, and penicillin/streptomycin (all from PAN Biotech) added. Cells were seeded into medium at a concentration of 1 × 105 cells/mL, cultured at 37 °C with 5% CO2, and split twice in a ratio of 1:5 per week. For cytochemistry, the cells were seeded at a concentration of 5 × 105 cells/mL in a 24-well culture plate on glass coverslips (Sigma Aldrich), and cultured for 48 h at 37 °C. Thereafter, cells were incubated with normal culture medium or medium containing test substances as optical probes and nanosensors for different times at 37 °C. Afterwards, the cells were fixed with 4% PFA, rinsed and 4,6-diamidino-2-phenylindole (DAPI, Abcam) was used for nuclear counterstaining.
9
Measuring Uric Acid with Gold Nanoparticles
10
Newborn Rat Vestibular Explant Culture
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Newborn rats (P0-P4) were decapitated, and temporal bones were placed in Leibovitz's L-15 medium. Ampullae were aseptically removed and care was taken to preserve the epithelium covering the ampullar crest. The crista ampullaris were stripped from the semi-circular duct to retain only the ampullae. Explants were then placed on 10 µL of Matrigel® (Corning, NY, USA) on 12-mm diameter laminin-coated (10 µg/mL) glass coverslips (Sigma-Aldrich, Saint-Louis, MO, U.S.A.). Structures were positioned so that the base of the sensory epithelium faced the coverslip. To solidify the matrix, samples were incubated at 37 • C for 30 min in a 95% O 2 / 5% CO 2 atmosphere at saturating humidity. Embedded explants were then covered with Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM-F12, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2% N-2 (Life Technologies, Carlsbad, California). Cultured ampullae were maintained at 37 • C under a humidified 5% CO 2 atmosphere, renewing half of the culture medium three times per week. After incubation for 24 h, explants sealed themselves to delimit a compartment filled with an endolymphlike fluid. The day of seeding was considered as 0 day in vitro (DIV).
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