Cell Navigator® CDy6 Mitosis Imaging Kit

Mitosis is the most defining stage of cell growth. The Cell Navigator® CDy6 Mitosis Imaging Kit is a useful tool for monitoring mitosis by visualizing lysosome dynamics. Long-term real-time visualization of mitosis had been a challenge due to the lack of photostable and low toxicity fluorescent probes. This CDy6 mitosis imaging kit uses a cell permeable lysosome dye, CDy6, which displays high sensitivity in an acidic environment and exhibit bright signal in lysosomes. During mitosis, lysosomes rapidly accumulate towards the nucleus, displaying a sharp increase in signal intensity that can be visualized in real-time. CDy6 does not interfere with the cell cycle and can stand repeated exposure for long-term imaging.

Example protocol

AT A GLANCE

Protocol summary

Prepare cells in a 96-well plate (100 µL/well)
Add 10X CDy6 working solution (10 µL/well)
Add test compounds
Incubate for the desired amount of time at 37 °C
Image with a fluorescence microscope using a Cy3/TRITC filter set

Important

Thaw all the kit components at room temperature before use.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. CDy6 stock solution (1000X)

Add 15 µL of DMSO into the vial of CDy6 (Component A) to make 1000X CDy6 stock solution.
Note Protect from light.

2. Nocodazole stock solution (1000X)

Add 50 µL of DMSO into the vial of Nocodazole (Component B) to make 1000X Nocodazole stock solution.

PREPARATION OF WORKING SOLUTION

1. CDy6 working solution (10X)

Add 1 µL of 1000X CDy6 solution in 100 µL of culture media or buffer of your choice and mix well.
Note 100 µL of 10X CDy6 working solution is enough for 10 tests in a 96-well plate format. Prepare enough 10X CDy6 working solution right before the experiment, and use promptly.

2. Optional: Nocodazole working solution (10X)

Prepare 10X Nocodazole working solution for positive control by mixing 1 µL of Nocodazole with 100 µL of culture media and mix well.

SAMPLE EXPERIMENTAL PROTOCOL

Plate 1000 to 40,000 cells/well (90 µL for 96-well plate, or 22.5 µL for 384-well plate) in a tissue culture microplate with clear bottom.
Add 10 µL/well (96-well plate) or 2.5 µL/well (384-well plate) of 10X CDy6 working solution to each well.
Add test compounds into the cells and incubate for a desired period of time (such as 24, 48 or 96 hours) in a 5% CO₂ incubator at 37 °C.
(Optional) For positive control, add 11 µL/well (96-well plate) or 3 µL/well (384-well plate) of 10X Nocodazole working solution to each well and incubate for 16 hours in a 5% CO₂ incubator at 37 °C. Mitotic cell population will be significantly enriched after the treatment.
Note Each cell line should be evaluated on an individual basis to determine the optimal cell density.
Image with a fluorescence microscope using a Cy3/TRITC filter set.

References

View all 50 references: Citation Explorer

[An acute myeloid leukemia case with concurrent 11q23 anomaly and D13S319 deficiency diagnosed by combined inter- and metaphase fluorescence in situ hybridization].
Authors: Zhang, Zhenhao and Wang, Yanfang and Wan, Wei and Ke, Xiaoyan
Journal: Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics (2020): 48-51

Cyclodextrin-Based Peptide Self-Assemblies (Spds) That Enhance Peptide-Based Fluorescence Imaging and Antimicrobial Efficacy.
Authors: Jiao, Jin-Biao and Wang, Guan-Zhen and Hu, Xi-Le and Zang, Yi and Maisonneuve, Stéphane and Sedgwick, Adam C and Sessler, Jonathan L and Xie, Juan and Li, Jia and He, Xiao-Peng and Tian, He
Journal: Journal of the American Chemical Society (2020): 1925-1932

In Vitro FRET- and Fluorescence-Based Assays to Study Protein Conformation and Protein-Protein Interactions in Mitosis.
Authors: Ems-McClung, Stephanie C and Walczak, Claire E
Journal: Methods in molecular biology (Clifton, N.J.) (2020): 93-122

Is in vivo and ex vivo irradiation equally reliable for individual Radiosensitivity testing by three colour fluorescence in situ hybridization?
Authors: Mayo, Theresa and Haderlein, Marlen and Schuster, Barbara and Wiesmüller, Anna and Hummel, Christian and Bachl, Maximilian and Schmidt, Manfred and Fietkau, Rainer and Distel, Luitpold
Journal: Radiation oncology (London, England) (2019): 2

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy.
Authors: Escorcia, Wilber and Shen, Kuo-Fang and Yuan, Ji-Ping and Forsburg, Susan L
Journal: Journal of visualized experiments : JoVE (2019)

Page updated on November 21, 2024

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