Glass bottomed 35 mm petri dishes

Manufactured by MatTek

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Glass-bottomed 35 mm Petri dishes are a type of laboratory equipment used for cell culture and other biological applications. They provide a transparent glass surface for observing and studying cells under a microscope.

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8 protocols using glass bottomed 35 mm petri dishes

Mitophagy Evaluation in H9c2 Cells

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To assay mitophagy (selective removal of damaged mitochondria by autophagosomes and lysosomes), H9c2 rat cardiomyoblasts (50 × 10⁴) were cultured in 35-mm glass-bottomed Petri dishes (MatTek, Ashland, MA, USA) with ZnO NPs (2.5, 5, 10 and 20 μg/cm²) for 6 h. To detect nuclei, mitochondria, or lysosomes, cells were pre-incubated with 0.4 µM Bis-Benzimide H 33342 trihydrochloride (Hoechst), 0.5 µM MitoTracker Green (MTG), and 0.5 µM LysoTracker Red (LTR), respectively, for 30 min at 37 °C in DMEM without phenol red. Epifluorescence images were taken with the EVOS FL (Thermo Fisher Scientific, Waltham, MA, USA) cell-imaging microscope at 60X magnification.

Mendoza-Milla C., Macías Macías F.I., Velázquez Delgado K.A., Herrera Rodríguez M.A., Colín-Val Z., Ramos-Godinez M.D., Cano-Martínez A., Vega-Miranda A., Robledo-Cadena D.X., Delgado-Buenrostro N.L., Chirino Y.I., Flores-Flores J.O, & López-Marure R. (2022). Zinc Oxide Nanoparticles Induce Toxicity in H9c2 Rat Cardiomyoblasts. International Journal of Molecular Sciences, 23(21), 12940.

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Imaging Mitochondrial and Lysosomal Dynamics

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COLO 205 and HCT 116 cells (3 × 10⁴/ml) were cultured in 35-mm glass-bottomed Petri dishes (MatTek; Ashland, MA, USA) in the presence of glucose and the different SCCAs. To reveal mitochondrial subcellular localization, cells were washed and pre-incubated with MitoTracker Green (MTG, 0.5 µM). To reveal lysosomes, autophagosomes, and autolysosomes, cells were pre-incubated with LysoTracker Red (LTR, 0.5 µM). Cells were loaded with both dyes for 20 min at 30°C in DMEM. Time series of confocal images were collected every 1 to 2 min with a Zeiss LSM 510 meta inverted laser scanning confocal microscope (Carl Zeiss; Oberkochen, Germany) using 63X oil 1.4 N.A. plan apochromat objective lens. LTR λ_excitation of 543 nm was provided by a helium/neon laser, and λ_emission of 560 nm was measured. MTG λ_excitation of 488 nm was provided by an argon laser, and λ_emission of 500–550 nm was followed. Laser excitation energy was attenuated 1,000-fold to minimize photobleaching and photodamage (41 (link)).

Rodríguez-Enríquez S., Robledo-Cadena D.X., Gallardo-Pérez J.C., Pacheco-Velázquez S.C., Vázquez C., Saavedra E., Vargas-Navarro J.L., Blanco-Carpintero B.A., Marín-Hernández Á., Jasso-Chávez R., Encalada R., Ruiz-Godoy L., Aguilar-Ponce J.L, & Moreno-Sánchez R. (2021). Acetate Promotes a Differential Energy Metabolic Response in Human HCT 116 and COLO 205 Colon Cancer Cells Impacting Cancer Cell Growth and Invasiveness. Frontiers in Oncology, 11, 697408.

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Transfection and Live-Cell Imaging Protocol

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Cells were plated in an antibiotic-free growth medium to either glass-bottomed 35 mm Petri dishes (MatTek) or eight-well chambers (LabTek). The following day, transfection was carried out using Lipofectamine 2000 or Lipofectamine 3000 (Invitrogen) following the manufacture’s recommendation. After incubation with the lipofectamine–DNA complex for ~5 h, the medium was replaced with a fresh growth medium and incubated overnight at 37 °C, 95% humidity, and 5% CO₂. Prior to imaging, the transfected cells were washed for 10 min three times with buffered saline solution (20 mM HEPES (pH 7.4), 135 mM NaCl, 5 mM KCl, 1 mM MgCl, 2.5 or 5.6 mM glucose, and 1 mg mL⁻¹ bovine serum albumin (Fisher Scientific)) and allowed to incubate in the dark at ambient temperature for at least 1 h. Cells were then imaged in the buffered saline solution.

Schmitt D.L., Cheng Y.J., Park J, & An S. (2016). Sequestration-Mediated Downregulation of de Novo Purine Biosynthesis by AMPK. ACS chemical biology, 11(7), 1917-1924.

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Culturing and Propagating Three Cell Lines

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Chinese hamster ovary cells (CHO-K1) and human epithelial kidney cells (HEK 293) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and propagated as recommended by the supplier. Bovine pulmonary artery endothelial cells (BPAE) were a kind gift of Dr. J. Catravas (Center for Bioelectrics, ODU, Norfolk, VA, USA). These three cell lines were propagated, respectively, in Ham’s F-12 K medium (Atlanta Biologicals, Norcross, GA, USA); EMEM with 1.5 g/L sodium bicarbonate, non-essential amino acids, L-glutamine and sodium pyruvate (Mediatech Cellgro, Herndon, VA, USA); and a low-glucose DMEM with 2.5 µg/mL amphotericin B (Thermo Scientific, Waltham, MA, USA). The media were supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA) and antibiotics (100 IU/mL penicillin and 0.1 mg/mL streptomycin, Gibco, Gaithersburg, MD, USA). Cells were grown in flasks at 37 °C, 5% CO2, and 1–2 days prior to experiments were passaged onto glass coverslips (BPAE and HEK) or into glass-bottomed 35-mm Petri dishes (MatTek, Ashland, MA, USA).

Bo W., Silkunas M., Mangalanathan U., Novickij V., Casciola M., Semenov I., Xiao S., Pakhomova O.N, & Pakhomov A.G. (2020). Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca2+ and Ba2+ Ions. International Journal of Molecular Sciences, 21(9), 3386.

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Measuring Cellular Elasticity via AFM

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Cells were cultured on glass-bottomed 35-mm Petri dishes (MatTek) and placed on the stage of an inverted optical microscope (Olympus IX70-S8F2) equipped with a microinbucator (HCMIS ALA Science) to hold the temperature at 37°C. A commercial stand-alone AFM (Bioscope, Veeco) adapted to the optical microscope and provided with a low-spring constant (0.03 nN/nm) AFM cantilever (Veeco) was used to assess the Young’s modulus (E) of single cells, following a protocol described in detail elsewhere (Alcaraz et al., 2003 (link), 2011 (link)). The spring constant of the cantilever was calibrated using the thermal fluctuations method (Roca-Cusachs et al., 2008 (link)). In brief, three standard force versus displacement curves (F vs. z) were recorded on the perinuclear region of each cell at moderate loading force (∼1 nN) and low speed (∼5 µm/s). The E of a single cell was computed by fitting a suitable contact elastic model to each F-versus-z curve and averaging it over the three recordings. The final E for a cell population in each experimental condition was calculated from at least nine measurements for each independent experiment (n ≥ 2).

Gabasa M., Duch P., Jorba I., Giménez A., Lugo R., Pavelescu I., Rodríguez-Pascual F., Molina-Molina M., Xaubet A., Pereda J, & Alcaraz J. (2017). Epithelial contribution to the profibrotic stiff microenvironment and myofibroblast population in lung fibrosis. Molecular Biology of the Cell, 28(26), 3741-3755.

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Transfection of Mammalian Cells

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Cells were plated in glass-bottomed 35 mm Petri dishes (MatTek) or 8-well chambers (LabTeK) in the absence of antibiotics. The next day, cells were transiently transfected with Lipofectamine 2000 (Invitrogen) or Xfect (Clontech) to express GFP-tagged proteins in HeLa and/or Hs578T cells. Following incubation with the Lipofectamine-DNA complexes for 5 h, cells were washed and subsequently incubated with fresh growth media in a HeraCell CO₂ incubator (37 °C, 5% CO₂, and 95% humidity) for ∼20 h. However, the Xfect-mediated transfection did not require removal of the Xfect-DNA complexes by washing. On the day of imaging, cells were washed for three 10 min incubations with Hank’s Balanced Salt Solution (HBSS; Invitrogen) or buffered saline solution (BSS: 20 mM HEPES [pH 7.4], 135 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 5.6 mM glucose), followed by ∼1–2 h incubation at ambient temperature. Note that we screened various transfection reagents with Hs578T cells to examine their relative transfection efficiency; other than Lipofectamine 2000 (Invitrogen) and Xfect (Clontech), those included Lipofectamine LTX and PLUS (Invitrogen), X-tremeGENE (Roche), TransPass D2 (New England Biolab), and Effectene (Qiagen).

Kyoung M., Russell S.J., Kohnhorst C.L., Esemoto N.N, & An S. (2015). Dynamic Architecture of the Purinosome Involved in Human De Novo Purine Biosynthesis. Biochemistry, 54(3), 870-880.

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Preparation of Hs578T cells for Transfection

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To prepare cells for transfection and subsequent imaging, Hs578T cells were gently removed from the culture flask by replacing the culture medium with Trypsin-EDTA solution (Corning, Cat# 25-053-Cl). Fresh, antibiotic-free growth media was subsequently used to harvest and resuspend the cells, followed by plating on glass-bottomed 35 mm Petri dishes (MatTek). When the confluency became ~70–90% on the following day, the cells were transfected with Lipofectamine 2000 (Invitrogen) using the Opti-MEM-I reduced serum media (Opti-MEM-I; Gibco, Cat# 11058). The Opti-MEM-I medium was then exchanged with the fresh antibiotic-free growth medium after a 5 h incubation (37 °C, 5% CO₂, and 95% humidity), followed by ~18–24 h incubation in the CO₂ incubator at 37 °C.

Jeon M., Kang H.W, & An S. (2018). A Mathematical Model for Enzyme Clustering in Glucose Metabolism. Scientific Reports, 8, 2696.

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Transient Transfection of HeLa Cells

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Parental HeLa (ATCC CCL-2) cells were acquired from the American Tissue Culture Collection (ATCC, Manassas, VA) and maintained in RPMI 1640 supplemented with 10% dialyzed FBS and 50 μg/mL gentamycin sulfate in a HeraCell CO₂ incubator (37°C, 5% CO₂, and 95% humidity). Briefly, HeLa cells were plated on either glass-bottomed 35 mm Petri dishes (MatTek) or 8-well chambers (LabTek) in RPMI 1640 supplemented with 10% dialyzed FBS without antibiotics. The following day, cells were transfected with PFK1-mEGFP using Lipofectamine 2000 (Invitrogen, Cat# 11668019) in Opti-MEM I (ThermoFisher, Cat# 11058–021) and allowed to incubate in a HeraCell CO₂ incubator (37°C, 5% CO₂, and 95% humidity). Approximately 5 hours later, the medium was exchanged for a fresh growth medium and incubated overnight in the CO₂ incubator.

Schmitt D.L., Dranchak P., Parajuli P., Blivis D., Voss T., Kohnhorst C.L., Kyoung M., Inglese J, & An S. (2023). High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells. PLOS ONE, 18(8), e0289707.

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