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| Overview |
Example protocol
AT A GLANCE
Protocol summary
- Grow cells as desired
- Add VSB405 loading solution and incubate for 45 minutes at RT
- Remove the VSB405 loading solution and wash cells with Voltage Assay Buffer I
- Add VSR555 loading solution and incubate for 15 minutes at RT
- Measure the response at Ex1/Em1 = 405/460 nm and Ex2/Em2 = 405/580 nm before and after addition of depolarizing stimulant
Important
Bring all the kit components at room temperature before starting the experiment.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.
Note Store the unused VSB405 stock solution at -20 °C in single use aliquots.
Note VSB405 stock solution is stable for at least one month at indicated storage conditions.
Note Store the unused VSR555 stock solution at -20 °C in single use aliquots.
Note VSR555 stock solution is stable for at least one month at indicated storage conditions.
1. VSB405 stock solution (100X)
Add 100 µL DMSO (Component F) into VSB405 (Component A) and mix well.Note Store the unused VSB405 stock solution at -20 °C in single use aliquots.
Note VSB405 stock solution is stable for at least one month at indicated storage conditions.
2. VSR555 stock solution (100X)
Add 100 µL DMSO (Component F) into VSR555 (Component C) and mix well.Note Store the unused VSR555 stock solution at -20 °C in single use aliquots.
Note VSR555 stock solution is stable for at least one month at indicated storage conditions.
PREPARATION OF WORKING SOLUTION
1. VSB405 loading solution
Add 10 µL 100X Pluronic® F127 (Component B) into 1 mL of Voltage Assay Buffer I (Component D), mix well. Add 10 µL VSB405 stock solution and mix well.Note VSB405 loading solution should not be stored and should be used promptly.
2. VSR555 loading solution
Add 10 µL VSR555 stock solution into 1 mL of Voltage Assay Buffer II (Component E) and mix well.Note VSR555 loading solution should not be stored and should be used promptly.
SAMPLE EXPERIMENTAL PROTOCOL
The following protocol can be used as a guideline and should be optimized according to the needs.
- Grow cells as desired.
- Remove the cell culture medium and add 100 µL Voltage Assay Buffer I (Component D).
- Immediately remove the Voltage Assay Buffer I.
Note It is not necessary to incubate or shack the cells with Voltage Assay Buffer I. - Add 100 µL VSB405 loading solution to each well and incubate at room temperature for 45 minutes, with plate covered and protected from light.
- Remove VSB405 loading solution and wash cells with 100 µL Voltage Assay Buffer I. Remove Voltage Assay Buffer I.
- Add 100 µL VSR555 loading solution to each well and incubate at room temperature for 15 minutes, with plate covered and protected from light.
- Read cells in plate reader at Ex1/Em1= 405/460 nm and Ex2/Em2= 405/580 nm. Plate reader will take readings in resting potential and then inject depolarization buffer such as high K buffer, before taking several more readings.
Note Test compounds should be added immediately after loading the cells with VSR555. Stimulants should be added while acquiring data.