Cell Navigator® Live Cell Tubulin Staining Kit

Imaging of tubulins in live HeLa cells. HeLa cells were co-labelled with Tubulite<sup>TM</sup> Red and DAPI (Cat# 17507) for 60 minutes in a 37 <sup>o</sup>C, 5% CO<sub>2</sub> incubator. The fluorescence image was taken with a fluorescence microscope (Cy5 filter set).

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Certificate of Origin	Download PDF
Intended use	Research Use Only (RUO)

Overview SDS Protocol

Cell Navigator® Live Cell Tubulin Staining Kit provides a robust method to fluorescently image tubulins in live cells with Tubulite™ Red. The probe is permeant to live cells, thus does not require cells to be fixated for imaging tubulins. It can be conveniently used for tracking tubulin polymerization process in live cells. Its red spectral wavelengths and good cell permeability make this probe readily to use with other colors such as GFP expressed cells or nuclear dyes such as DAPI. The neutral Tubulite™ Red readily passes through the plasma membranes of live cells. Once inside the cells, the lipophilic blocking group is cleaved by esterases, resulting into a negatively charged product, which is well retained inside the cells.

Platform

Fluorescence microscope

Excitation	Cy5 filter set
Emission	Cy5 filter set
Recommended plate	Black wall/clear bottom

Components

Example protocol

AT A GLANCE

Protocol Summary

Prepare cells with test compounds at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL
Prepare and add Tubulite^TM Red working solution to cells
Incubate at 37 °C for 30 to 60 minutes
Read fluorescence intensity with Cy5 filter set

Important 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.
Tubulite™ Red does not stain formaldehyde fixed cells. Cells can not be fixed after staining with Tubulite™ Red as fixation alters the structure of micotubules.

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.

Tubulite^TM Red stock solution (500X)

Add 25 µL DMSO (Component D) into the vial of Tubulite^TM Red (Component A), and mix well. Note: Aliquot and stored the unused Tubulite^TM Red stock solution at -20 °C. Avoid repeated freeze/thaw cycles.

PREPARATION OF WORKING SOLUTION

Tubulite^TM Red working solution (1X)

Add 2.5 µL of Tubulite^TM 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^TM 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^TM 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 cells with Assay Buffer with 2.5 mM probenecid (diluted from Component C) and monitor the fluorescence intensity with fluorescence microscope using Cy5 filter set.

Images

Figure 1. Imaging of tubulins in live HeLa cells. HeLa cells were co-labelled with Tubulite^TM Red and DAPI (Cat# 17507) for 60 minutes in a 37 ^oC, 5% CO₂ incubator. The fluorescence image was taken with a fluorescence microscope (Cy5 filter set).

Citations

View all 1 citations: Citation Explorer

Self-assembly of CXCR4 antagonist peptide--docetaxel conjugates for breast tumor multi-organ metastasis inhibition
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)