A431 cells (each 2 × 105 cells/2 mL) were seeded onto a 35‐mm glass dish (Iwaki) and incubated in MEM containing 10% FBS for 24 h at 37 °C under 5% CO2. After complete adhesion, the cells were then treated with each EV sample (200 μL per well) in MEM at different pH conditions (pH 5, 6, or 7), with or without 10% FBS. The cells were stained with Hoechst 33342 dye (Invitrogen; 5 μg·mL−1) for 15 min at 37 °C, prior to cell washing. The cells were then washed with fresh cell culture medium and analyzed using a FV1200 confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a 40× objective without cell fixation.
35 mm glass dish
Manufactured by Iwaki
Sourced in Japan
The 35-mm glass dish is a laboratory equipment item used for cell culture and other scientific applications. It provides a contained environment for conducting experiments or maintaining samples. The dish is made of durable glass material and has a circular design with a flat bottom.
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9 protocols using 35 mm glass dish
1
Visualizing EV Uptake in A431 Cells
2
CD63-GFP HeLa Cells pH Response
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CD63‐GFP stably expressing HeLa cells (each 2 × 105 cells/2 mL) were seeded onto a 35‐mm glass dish (Iwaki) and incubated in α‐MEM containing 10% FBS for 24 h at 37 °C under 5% CO2. After complete adhesion, the cells were treated with α‐MEM at different pH conditions, containing 10% FBS (pH 5 or 7; 2 mL per well) for 24 h at 37 °C under 5% CO2. The cells were then washed with fresh cell culture medium and analyzed using a FV1200 confocal laser scanning microscope (Olympus) equipped with a 40× objective without cell fixation.
3
Fluorescent Polymer Transfection into HeLa Cells
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HeLa cells were cultured on a 35-mm glass dish (glass 27 ϕ, Iwaki, Japan) in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Kyoto, Japan) supplemented with FBS (Hyclone SH30910.03, Lot# AVB64834, Thermo Scientific, Japan) at 37°C with 5% CO2. For loading of cell permeable FPT into cells, DMEM medium was removed from a dish containing cells at 30 to 50% confluency, and cells were rinsed with 1 mL of 1×PBS (10×PBS contains 28.9 g of Na2HPO4–12H2O, 2.0 g of KH2PO4, 80.0 g of NaCl, and 2.0 g of KCl in 1 L solution). Then PBS was replaced with 1 mL of cell-permeable FPT at appropriate concentration in 5 w/v% glucose in water (for 0.01 w/v % of FPT, 2 µL of 5 w/v% FPT stock solution in water was diluted in 998 µL of 5 w/v% glucose in water. 5 w/v% stock solution in water was prepared and incubated at 4°C at least overnight before use to obtain full extension of the polymer). The dish was incubated at either 4°C or 25°C for 5 to 20 min without CO2 supply. After incubation, FPT solution was removed, and cells were rinsed with 1 mL of 1×PBS three times. 2 mL of phenol red-free DMEM medium (Gibco No. 21063, Life Technologies Japan, Tokyo, Japan) was added to the dish before imaging.
4
Visualizing Cellular Dynamics with EV Treatment
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CHO-K1 cells (2 × 105 cells, 2 mL) were plated onto a 35-mm glass dish (Iwaki, Tokyo, Japan) and incubated in Ham’s F-12 containing 10% FBS for 24 h at 37 °C under 5% CO2. After complete adhesion, the cells were washed with cell culture medium containing 10% FBS and treated with each EV sample (100 μl/well). The cells were stained with Hoechst 33342 dye (Invitrogen; 5 μg/ml) for 15 min at 37 °C before cell washing. The cells were then washed with fresh cell culture medium and analysed using a FV1200 confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a 40x objective without cell fixation.
To visualize the lamellipodia formation of cellular actin, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and washed with PBS after the cells were treated with each EV sample (100 μl/well). The cells were then treated with 0.1% Triton X-100 (100 μl/well in PBS) at room temperature for 5 min and again washed with PBS. Cellular F-actin was stained with rhodamine-phalloidin (2.5 μl (300 units) in PBS (97.5 μl)/well) (Molecular Probes) for 20 min at 4 °C, and the cells were washed with PBS before analysis using a FV1200 confocal laser scanning microscope (Olympus) equipped with a 40× objective.
To visualize the lamellipodia formation of cellular actin, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and washed with PBS after the cells were treated with each EV sample (100 μl/well). The cells were then treated with 0.1% Triton X-100 (100 μl/well in PBS) at room temperature for 5 min and again washed with PBS. Cellular F-actin was stained with rhodamine-phalloidin (2.5 μl (300 units) in PBS (97.5 μl)/well) (Molecular Probes) for 20 min at 4 °C, and the cells were washed with PBS before analysis using a FV1200 confocal laser scanning microscope (Olympus) equipped with a 40× objective.
5
Visualizing Actin Cytoskeleton Dynamics
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HeLa cells (2 × 105 cells, 2 ml) were plated onto a 35-mm glass dish (Iwaki) and incubated in cell culture medium (α-MEM) containing 10% FBS for 24 h at 37 °C under 5% CO2. After complete adhesion, the cells were washed with cell culture medium containing 10% FBS and treated with each EV sample (100 μl/well) for 20 min at 37 °C. After removal of the medium, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and washed with PBS. The cells were then treated with 0.1% Triton X-100 (100 μl/well in PBS) at room temperature for 5 min and again washed with PBS. Cellular F-actin was stained with rhodamine-phalloidin (Molecular Probes) for 20 min at 4 °C, and the cells were washed with PBS prior to analysis using a FV1200 confocal laser scanning microscope (Olympus) equipped with a 40× objective.
6
Exosomal Uptake in Cell Cultures
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Cells (2 × 105 cells, 2 ml) were plated on a 35 mm glass dish (Iwaki, Tokyo, Japan) and incubated in cell culture medium (HeLa cells, α-MEM; CHO-K1 cells, Ham’s F-12) containing 10% FBS for 24 h at 37 °C under 5% CO2. After complete adhesion, the cells were washed with cell culture medium containing 10% FBS and treated with exosomal samples (200 μl/well). The cells were then washed with fresh cell culture medium and analysed using a FV1200 confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a ×40 objective. To stain endosomes, HeLa cells were pre-stained with DiD (Invitrogen, 2.5% (v/v)) in 10% FBS containing α-MEM (200 μl/well) at 4 °C for 5 min. The cells were then washed with fresh cell culture medium, and treated with CD63-GFP-exosomes (20 μg/ml) with the combined treatment with Lipofectamine LTX (2% (v/v)) at 37 °C for 6 h, prior to observation by confocal microscope. To prevent endosomal pH-reduction, NH4Cl (50 mM) was added in exosomal samples as previously reported.20 (link)
7
Imaging Spinal Motor Neuron Axon Navigation
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MOspATG1, MOspATG2 and MOscr-injected Tg(Hb9:GFP) transgenic embryos were anesthetised at 40 hpf with tricaine and embedded in 0.8% low melting agarose in a 35 mm glass dish (Iwaki). Time-lapse videomicroscopy recordings of sMN axon navigation were carried out using a Leica DMI 6000B inverted spinning-disk microscope with a 40×/1.25 NA immersion objective over a period of 32 h. Z-stacks of 1 µm were taken over a 70 µm depth every 8 min and compiled into time-lapse movies or figure panels.
8
Cellular Uptake of Fluorescent nsEVs
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HeLa cells (2 × 105 cells, 2 ml) were plated on a 35 mm glass dish (Iwaki, Tokyo, Japan) and incubated in the cell culture medium. After complete adhesion, cells were washed with culture medium and treated for 24 h with nsEV samples (25 µg/ml), either treated with electroporation or sodium carbonate buffer protocol to load dextran-FITC. Cells were then washed with fresh cell culture medium and analyzed using an FV1200 confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a 40 × magnification objective [8 (link)].
9
Spinal Motor Axon Outgrowth Analysis
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Control and MO p60Kat -injected Tg(Hb9:GFP) embryos were anesthetised with E3 medium containing tricaine and embedded in 0.8% low-melting agarose in a 35-mm glass dish (Iwaki). Time-lapse videomicroscopy recording of spinal motor axon outgrowth was carried out at 28°C in E3 medium (supplemented with tricaine) using a preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted January 20, 2022. ; https://doi.org/10.1101/2022.01.20.477127 doi: bioRxiv preprint 12 Leica DMI 6000B inverted spinning-disk microscope with a 40x immersion objective (NA 1.4) and a 491-nm 100-mW Cobolt calypso laser over 30 hours. Embryos immobilised at 22 hpf were filmed with a 20x immersion objective over 48 hours. In both cases, z-stacks of 80-µm-thick sections were acquired every 8 minutes with a step size of 1 µm using an EMCCD camera (Photometrics Quantem 512 SC) and the Metamorph software (Molecular Devices) and compiled into time-lapse movies.
The copyright holder for this this version posted January 20, 2022. ; https://doi.org/10.1101/2022.01.20.477127 doi: bioRxiv preprint 12 Leica DMI 6000B inverted spinning-disk microscope with a 40x immersion objective (NA 1.4) and a 491-nm 100-mW Cobolt calypso laser over 30 hours. Embryos immobilised at 22 hpf were filmed with a 20x immersion objective over 48 hours. In both cases, z-stacks of 80-µm-thick sections were acquired every 8 minutes with a step size of 1 µm using an EMCCD camera (Photometrics Quantem 512 SC) and the Metamorph software (Molecular Devices) and compiled into time-lapse movies.
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