Tubulite™ Green *Cell-Permeable*

1. Prepare cells with test compounds at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL

2. Prepare and add Tublite™ Green working solution to cells

3. Incubate at 37°C for 60 minutes.

4. Use a fluorescence microscope with a FITC filter set to measure fluorescence intensity.

   Note: This protocol is intended as a general guide and should be adjusted to meet your specific requirements.

1. For each sample, prepare cells in 1 mL of warm medium or buffer at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL.

   Note: Each cell line should be individually assessed to determine its optimal cell density.

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. Add 100 µL of DMSO (not provided) to the Tublite™ Green vial and mix thoroughly.

   Note: Aliquot and store any unused Tublite™ Green stock solution at -20°C. Avoid multiple freeze-thaw cycles.

1. To prepare a 1X Tubulite™ Green working solution, add 10 µL of Tublite™ Green stock solution and 1 mM ReadiUse™ Probenecid (AAT Cat# 20061, not provided) to 1 mL of HHBS [Hanks' Buffer with 20 mM Hepes] (AAT Cat# 20011, not provided) or your preferred buffer. Mix thoroughly.

   Note: For best results, we recommend preparing a fresh Tublite™ Green working solution each time you use it. The working solution remains stable for a few hours.

   Note: The calculations above assume that 100 µL of working solution will be used for each slide or sample. Please adjust the concentrations of Tublite™ Green and ReadiUse™ probenecid to match your specific staining and cell conditions.

1. Prepare cell samples as needed.

2. Remove the cell growth medium, then wash the cells with PBS (not included) or another buffer of your choice.

3. Add 100 µL of Tublite™ Green working solution to the cells, then place them in a 37°C incubator for 60 minutes.

   Note: The incubation time will vary based on the specific cell type and cell concentration. Be sure to adjust the incubation time to best suit each experiment.

4. Remove the working solution and wash the cells twice with PBS or a preferred buffer.

5. Cover the cells with HHBS (or a buffer of your choice) containing 1 mM probenecid. Then, use a fluorescence microscope equipped with a FITC filter set to monitor the fluorescence intensity.