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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).
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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Catalog Number23170
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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Intended useResearch Use Only (RUO)

OverviewpdfSDSpdfProtocol


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

ExcitationCy5 filter set
EmissionCy5 filter set
Recommended plateBlack wall/clear bottom

Components


Component A: Tubulite™ Red1 vial
Component B: Assay Buffer1 bottle (20 mL)
Component C: 25 mM ReadiUse™ probenecid (10X)1 bottle (10 mL)
Component D: DMSO1 vial (100 µL)

Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare cells with test compounds at a density of 5 × 105 to 1 × 106 cells/mL
  2. Prepare and add TubuliteTM Red working solution to cells
  3. Incubate at 37 °C for 30 to 60 minutes
  4. 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.

TubuliteTM Red stock solution (500X)
Add 25 µL DMSO (Component D) into the vial of TubuliteTM Red (Component A), and mix well. Note: Aliquot and stored the unused TubuliteTM Red stock solution at -20 °C. Avoid repeated freeze/thaw cycles.

PREPARATION OF WORKING SOLUTION

TubuliteTM Red working solution (1X)
Add 2.5 µL of TubuliteTM 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 TubuliteTM Red working solution fresh for every use. The working solution is stable for several hours.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Prepare cell samples as per need.
  2. Remove the cell growth medium and wash cells with PBS (Not provided) or any other buffer of your choice. (Optional).
  3. Add 100 µL TubuliteTM 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.
  4. 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).
  5. 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.