In this study, in addition to HeLa cells from human (Figure S1 ), COS7 cells from monkey, which are suitable for microinjection, were used. The miRNA and target mRNA sequences examined are conserved among mammals including these species.53 (link) COS7 cells and HeLa cells (Riken, Wako, Japan) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum supplemented with penicillin–streptomycin, L-glutamine, sodium pyruvate, and nonessential amino acids at 37°C in 5% CO2. For live-cell imaging, cells were cultured in 35-mm glass-bottomed dishes (AGC Techno Glass, Yoshida, Japan) and the medium was replaced by phenol red-free culture medium containing HEPES buffer (2 mL) before live-cell imaging. All solutions were from Thermo Fisher Scientific.
35 mm glass bottomed dishes
Manufactured by AGC Techno Glass
Sourced in Japan
35-mm glass-bottomed dishes are circular, transparent cell culture vessels made of borosilicate glass. They provide a flat, rigid, and optically clear surface for microscopic observation of cells and tissues.
Automatically generated - may contain errors
4 protocols using 35 mm glass bottomed dishes
1
Live-cell Imaging of miRNA-mRNA Interactions
2
Visualizing NFAT Translocation in BHK-21 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BHK-21 cells were cultured in Dulbecco Modified Eagle Medium, high glucose containing 10% fetal bovine serum (FBS), 5 mg / ml puromycin (Nacalai Tesque, Kyoto Japan), 100 IU / mL penicillin, and 100 µg / ml streptomycin. The cells were placed onto 35-mm glass-bottomed dishes (AGC Techno Glass, Shizuoka, Japan). The cells were treated with pyrogallol (100 µM) for 24 h before stimulation with 1 µM of ionomycin. After stimulation, the cells were washed once with Ca 2+ -free PBS (PBS(-)) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min at 4°C. Nuclear stain was conducted by the addition of 0.25 µg / ml of 4',6-diamidino-2-phenylindole (DAPI) in PBS(-) containing 1% BSA and 0.1% Tween 20 for 10 min. The subcellular localization of EGFP-tagged NFAT was determined using a confocal laser microscope (LSM510 ; Carl Zeiss, Oberkochen, Germany).
3
Quantifying Mitochondrial Morphology Changes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded on 35-mm glass-bottomed dishes (AGC Techno Glass, Shizuoka, Japan) and cultured overnight. At the times indicated after x-irradiation, the cells were incubated with serum-free medium containing 300 nM MitoTracker Green FM for 30 min at 37°C. After two washes with serum-free medium, fresh growth medium was supplied. Fluorescence images of live cells were obtained using an LSM700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) at 37°C in 5% CO2. To quantify mitochondrial morphology, a cell was judged to have fragmented mitochondria if >75% of the mitochondria visible in the cell were punctate or circular and fused if <25% of the mitochondria were punctate or circular. The fraction of fused mitochondria was subdivided into highly connected and tubular, depending on the length of mitochondrial filaments (“highly connected” mitochondria appear more elongated than “tubular” mitochondria). If cells presented mitochondria with mixed morphologies, we classified them as intermediate. Representative images of the different mitochondrial morphologies in each category are shown in Supplemental Figure S1. In one experiment, >50 cells/condition were randomly chosen and classified as described, and the percentage of each category was calculated. Experiments were repeated three times.
4
Mitochondrial Dynamics in Irradiated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded on 35-mm glass-bottomed dishes (AGC Techno Glass, Shizuoka, Japan) and cultured overnight. At the times indicated after X-irradiation, the cells were incubated with serum-free medium containing 100 nM MitoTracker Green FM for 30 min at 37°C. After two washes with serum-free medium, fresh growth medium was supplied. Fluorescence images of live cells were obtained using an LSM700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) at 37°C in 5% CO2. The quantitative analysis of mitochondrial morphologies was performed as described previously [13] . Representative images of the different mitochondrial morphologies in each category are shown in Fig. S1. In each experiment, >50 cells/condition were randomly chosen and classified, and the percentage of each category was calculated.
Experiments were repeated three times.
Experiments were repeated three times.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!