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Cell Navigator® Live Cell Tubulin Staining Kit
The Cell Navigator® Live Cell Tubulin Staining Kit provides a robust method for the fluorescent visualization of tubulins in live cells using Tubulite™ Deep Red. This cell-permeable probe enables live-cell imaging of tubulin dynamics without requiring fixation, facilitating real-time monitoring of tubulin polymerization. The deep red fluorescence of Tubulite™ Deep Red and efficient cellular permeability allows for multiplexing with other fluorescent labels, including GFP and nuclear stains such as DAPI. Once Tubulite™ Deep Red traverses the plasma membrane, cellular esterases hydrolyze the lipophilic blocking group, yielding a charged, membrane-impermeant product that remains well-retained within the cell, enabling sustained and stable imaging of intracellular tubulin structures.
Example protocol
AT A GLANCE
Prepare cells with test compounds at a density of 5 × 105 to 1 × 106 cells/mL
Prepare and add Tubulite Deep™ Red working solution to cells
Incubate at 37 °C for 30 to 60 minutes
Read fluorescence intensity with Cy5 filter set
Thaw one of each kit component at room temperature before starting the experiment.
Note: This protocol only provides a guideline, and should be modified according to your specific needs.
Note: Tubulite™ Deep Red does not stain formaldehyde-fixed cells. Cells can not be fixed after staining with Tubulite™ Deep Red as fixation alters the structure of microtubules.
CELL PREPARATION
For guidelines on cell sample preparation, please visit:
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
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
Add 25 µL DMSO (Component D) into the vial of Tubulite™ Deep Red (Component A), and mix well.
Note: Aliquot and store the unused Tubulite™ Deep Red stock solution at -20 °C. Avoid repeated freeze/thaw cycles.
PREPARATION OF WORKING SOLUTION
Add 2.5 µL of Tubulite™ Deep Red stock solution stock solution and 100 µL 25 mM ReadiUse™ probenecid (Component D) into 1 mL of Assay Buffer (Component B) or buffer of your choice, and mix well.
Note: We recommend making Tubulite™ Deep Red working solution fresh for every use. The working solution is stable for several hours.
SAMPLE EXPERIMENTAL PROTOCOL
Prepare cell samples as per need.
Remove the cell growth medium and wash cells with PBS (Not provided) or any other buffer of your choice. (Optional).
Add 100 µL Tubulite™ Deep Red working solution and incubate them at 37 °C incubator for 30 to 60 minutes.
Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
Remove the working solution and wash cells twice with PBS or any other buffer of your choice with 2.5 mM probenecid (diluted from Component C).
Cover the cells with an Assay Buffer containing 2.5 mM probenecid (prepared by diluting Component C), and then monitor fluorescence intensity using a fluorescence microscope with a Cy5 filter set.
Spectrum
Citations
Authors: Lin, Hung-Yu and Tsai, Tsen-Ni and Hsu, Kai-Cheng and Hsu, Yu-Ming and Chiang, Lin-Chien and El-Shazly, Mohamed and Chang, Ken-Ming and Lin, Yu-Hsuan and Tu, Shang-Yi and Lin, Tony Eight and others,
Journal: Marine Drugs (2024): 323
Authors: Li, Chen and Lang, Jiayan and Wang, Yazhou and Cheng, Zhaoxia and Zu, Mali and Li, Fenfen and Sun, Jingyi and Deng, Yating and Ji, Tianjiao and Nie, Guangjun and others,
Journal: Acta Pharmaceutica Sinica B (2023)
Certificate of origin