To monitor peptide internalization, 1 mL of HeLa cell suspension (5 × 104 cells) was seeded in a 35-mm glass-bottomed microwell dish (MatTek) and cultured overnight. Cells were gently washed with DPBS twice and treated with 5 μM FAM-labeled peptide in phenol red-free DMEM containing 1% FBS for 37 °C in the presence of 5% CO2. After 2 h of incubation, the medium was removed, and the cells were gently washed with DPBS twice and phenol red-free DMEM was added. The cells were imaged on a Nikon A1R live-cell confocal microscope equipped with 100X oil objective in a heated chamber at 37 °C and 5% CO2. Images were captured under the same parameters. NIS-Elements AR was used to denoise the images and add scale bars. Images presented in this report were generated using the instruments and services at the Campus Microscopy and Imaging Facility, The Ohio State University.
35 mm glass bottomed microwell dish
Manufactured by MatTek
The 35-mm glass-bottomed microwell dish is a laboratory equipment item designed for cell culture applications. It features a glass bottom that allows for high-quality microscopic imaging of cells grown in the dish. The dish has a standard 35-mm diameter and is constructed with a durable material suitable for cell culture purposes.
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Lab products found in correlation
A1r live cell confocal microscope, by Nikon (1 mentions)
Plan apochromat 63 1.4 na objective, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Csu x1, by Zeiss (1 mentions)
Csu x1 spinning disc, by Yokogawa (1 mentions)
Metamorph premier, by Molecular Devices (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
5 protocols using 35 mm glass bottomed microwell dish
1
Monitoring Peptide Internalization in HeLa Cells
2
Live-cell Confocal Imaging Protocol
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Cells were imaged in DMEM without phenol red but with supplements, including 20 mM Hepes, pH 7.4. Transfection and imaging were performed in a 35-mm glass-bottomed microwell dish (MatTek) or glass coverslips. A Zeiss LSM710 or LSM800 confocal laser-scanning microscope was used (Carl Zeiss MicroImaging). Fluorescence emissions resulting from 405-nm excitation for CFP, 488-nm excitation for GFP, and 543-nm excitation for mCherry were detected using filter sets supplied by the manufacturer. The confocal and time-lapse images were captured using a Plan-Apochromat 63× 1.4-NA objective (Carl Zeiss MicroImaging). The temperature on the microscope stage was held stable during time-lapse sessions using an electronic temperature-controlled airstream incubator. Images and videos were generated and analyzed using the Zeiss LSM Zen software and National Institutes of Health Image and ImageJ software (W. Rasband, National Institutes of Health, Bethesda, MD). High-frequency images were captured using a Nikon Ti microscope equipped with a Yokogawa CSU X-1 spinning disc, controlled by Andor IQ2 software, or a spinning disk confocal unit (Yokogawa Electric; CSU-X1) attached to an Axio Observer Z1 microscope (Carl Zeiss).
3
Peptide Internalization in H1299 Cells
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A total of 1
mL of H1299 cell suspension (∼1.5 × 104 cells)
was seeded in a 35 mm glass-bottomed microwell dish (MatTek) and cultured
overnight. Cells were gently washed with PBS twice and treated with
FITC-labeled peptide (5 μM) in phenol-red free and HEPES supplemented
RPMI containing 10% FBS at 37 °C for 2 h in the presence of 5%
CO2. After removal of the medium, the cells were gently
washed with DPBS twice and imaged on a Visitech Infinity 3 Hawk 2D-array
live cell imaging confocal microscope equipped with 60× oil objective.
Data were analyzed using MetaMorph Premier (Molecular Devices).
mL of H1299 cell suspension (∼1.5 × 104 cells)
was seeded in a 35 mm glass-bottomed microwell dish (MatTek) and cultured
overnight. Cells were gently washed with PBS twice and treated with
FITC-labeled peptide (5 μM) in phenol-red free and HEPES supplemented
RPMI containing 10% FBS at 37 °C for 2 h in the presence of 5%
CO2. After removal of the medium, the cells were gently
washed with DPBS twice and imaged on a Visitech Infinity 3 Hawk 2D-array
live cell imaging confocal microscope equipped with 60× oil objective.
Data were analyzed using MetaMorph Premier (Molecular Devices).
4
Visualizing Rho-/FITC-Labelled Peptide Uptake in HeLa Cells
5
Preparation of Rat Hippocampal Neuronal Cultures
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Neuronal cultures were prepared from E18 rat hippocampi as described [5] (link). All experiments were performed in accordance with Institutional Animal Care and Use Committee guidelines and regulations. Rats were euthanized by CO2 inhalation in a chamber. CO2 flow was maintained until the rat was deeply anesthetized (verified via toe-pinch) and in CO2 narcosis. Death was confirmed by decapitation and embryos removed.
Hippocampi from all pups in one litter were combined and thus contained male and female animals. Cells were plated on poly-l-lysine-coated coverslips and incubated with plating medium containing DMEM with 10% horse serum. For live-imaging use, neurons were plated on a 35-mm glass-bottomed microwell dish (MatTek). After 4 h, the plating medium was removed and replaced with serum-free medium supplemented with B27 (Thermo Fisher Scientific), and neurons were cultured for 7-10 DIV for experimental use.
Transfections were performed with Lipofectamine 2000 (Invitrogen).
Hippocampi from all pups in one litter were combined and thus contained male and female animals. Cells were plated on poly-l-lysine-coated coverslips and incubated with plating medium containing DMEM with 10% horse serum. For live-imaging use, neurons were plated on a 35-mm glass-bottomed microwell dish (MatTek). After 4 h, the plating medium was removed and replaced with serum-free medium supplemented with B27 (Thermo Fisher Scientific), and neurons were cultured for 7-10 DIV for experimental use.
Transfections were performed with Lipofectamine 2000 (Invitrogen).
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