A. Reagent Preparation:
Prepare Pluronic F-127 stock solution: Weigh 100mg of Pluronic F-127 powder (Catalog Number: GB30090) and dissolve it in 500ul of DMSO to make a 20% (W/V) DMSO stock solution. The dissolution process should be carried out at 40-50°C for 20-30 minutes. Store the solution at room temperature, do not refrigerate. If crystals form, you can reheat to dissolve them, which will not affect its use.
HHBS Buffer (1X Hank's Balanced Salt Solution with 20mM HEPES buffer, pH 7.2-7.4), or other physiological buffer solutions.
B. Procedure:
Dissolve Cal-520 AM in anhydrous DMSO to prepare a stock solution of 2-5mM, or thaw a pre-made Cal-520 AM stock solution at room temperature. For example, if preparing a 4mM stock solution, add 11.3ul of anhydrous DMSO to 50ug of Cal-520 AM.
Dilute the Cal-520 AM+DMSO stock solution with HHBS or other physiological buffer to create a staining working solution containing 0.04% Pluronic F-127 and 10-20uM of Cal-520 AM.
[Note 1] Add a certain amount of 20% Pluronic F-127 solution to the Cal-520 AM+DMSO stock solution to achieve a final Pluronic F-127 concentration of approximately 0.04%. Pluronic F-127 prevents AM dyes from aggregating in the solution and facilitates better probe entry into cells. However, Pluronic F-127 may reduce the stability of the AM probe, so it is recommended to add it only when preparing the working solution and not for long-term storage of the stock solution.
[Note 2] The recommended loading concentration of Cal-520 AM for most cell types is 4-5μM. The specific concentration should be optimized based on experimental requirements. To avoid cytotoxicity due to overloading, it is advisable to use the lowest probe concentration that provides effective results.
[Note 3] The Cal-520 AM working solution should be prepared fresh and used immediately, avoiding repeated freeze-thaw cycles.
[Optional] If cells contain organic anion transporters (e.g., CHO cells), probenecid (1-2mM) may be needed in the staining working solution (resulting in a final concentration of 0.5-1mM) to reduce probe leakage.
Add an equal volume of the staining working solution to the cell culture wells and incubate at 37°C for 60-90 minutes. Then, incubate at room temperature for an additional 30 minutes.
[Note 1] Some cell lines may require longer than 2 hours for optimal probe loading.
Remove the staining working solution and wash the cells 1-2 times with HHBS or other physiological buffer (if necessary, use a buffer containing transporter inhibitors such as 1mM probenecid) to remove any residual probe.
Subsequently, in HHBS or other physiological buffer (if necessary, use a buffer containing transporter inhibitors such as 1mM probenecid), detect fluorescence using excitation/emission 492/514 nm.