Fluo-8 AM, Cell Permeant Calcium Ion Fluorescence Probe

Fluo-8 AM, Cell Permeant Calcium Ion Fluorescence Probe

Product Introduction

Fluo-8（Fluo-2 Medium Affinity）， It is currently the brightest visible light excitation wavelength Ca2+fluorescent probe, with fluorescence intensity twice as strong as Fluo-4 and four times stronger than Fluo-3. Fluo-8 has moderate Ca2+affinity, almost close to Fluo-3，Fluo-4（Fluo-3: Kd=0.4μM、Fluo-4: Kd=0.36μM、Fluo-4: Kd= 0.389μM）， It is almost identical to Fluo-3 and Fluo-4 in terms of usage. Fluo-8 not only maintains the convenient spectral detection characteristics of Fluo-3 and Fluo-4, but also has a maximum excitation wavelength of 490nm and a maximum emission wavelength of 514nm after binding with Ca2+. It also has improved cell loading and Ca2+response capabilities. Fluo-8 loading has weak temperature dependence and stains well at room temperature or 37 ℃ incubation, unlike Fluo-3 and Fluo-4 which strictly require incubation at 37 ℃. This feature makes it more suitable for high-throughput screening experiments in HTS. Fluo-8 has a wide range of applications and is compatible with many cell lines and targeted samples, without interference from ligand or target signals.

Fluo-8 can detect changes in intracellular calcium ion levels through laser confocal microscopy, flow cytometry, fluorescence enzyme-linked immunosorbent assay, fluorescence microscopy, and other methods. Fluo-8, AM is an acetyl methyl derivative of Fluo-8, which has cell membrane permeability and can easily enter cells with simple cultivation. Once inside the cell, Fluo-8 is cleaved by its lactonase to produce non membrane permeable Fluo-8, which then remains inside the cell to perform its corresponding physiological functions.

This product is in the form of freeze-dried powder in small packages of 50ug. When used for detecting intracellular Ca2+levels, the recommended concentration is 1-5uM.

Fluo-8 AM, Cell Permeant Calcium Ion Fluorescence Probe

Basic characteristics

Synonyms：Fluo-8, AM

molecular formula：C50H50N2O23

molecular weight：1046.9316

solubilityDissolved in DMSO (1-5 mM)

Ex/Em490/514nm (Ca2+binding);

Storage conditionsStore at -20 ℃ dry for 1 year.

Fluo-8 AM, Cell Permeant Calcium Ion Fluorescence Probe

Usage

Preparation of reagent A

1) Preparation of Pluronic F-127 mother liquor: Weigh 100mg of Pluronic F-127 powder (item number: Cat No. FS0432) and add 500ul of DMSO to prepare a 20% (W/V) mother liquor. The dissolution process requires heating at 40-50 ℃ for 20-30 minutes, and the solution should be stored at room temperature without refrigeration. If there is crystal precipitation, it can be reheated and dissolved without affecting its use.

2）HHBS BUFFER(1X Hank’s Balanced Salt Solution with 20mM HEPES buffer,pH7.3) Or other physiological buffer solutions

B Operation steps

1) Dissolve Fluo-8 and AM in anhydrous DMSO to prepare a storage solution of 1-5mM, or take the prepared Fluo-8 and AM storage solution back to room temperature. (For example, if preparing a 4mM mother liquor, 11.9ul anhydrous DMSO needs to be added to 50ug Fluo-8, AM).

2) Dilute the working solution with HHBS or other physiological buffer Fluo-8, AM+DMSO storage solution to 1-10 uM, and add an appropriate amount of 20% Pluronic F-127 solution in advance to achieve a final concentration of 0.02%.

[Note (1)]: The recommended loading concentration for Fluo-8 and AM in most cells is 4-5uM, and the specific usage concentration needs to be optimized according to experimental requirements. To avoid cell toxicity caused by overloading, it is recommended to use the lowest possible probe concentration based on the results obtained.

[Note (2)]: Fluo-8 and AM working fluids should be prepared and used immediately to avoid repeated freezing.

【 Note (3) 】: Pluronic F-127 can prevent Fluo-8 and AM from aggregating in solution and promote better entry of probes into cells. However, Pluronic F-127 can reduce the stability of Fluo-8 and AM, so it is only recommended to add it when preparing the working fluid and not to add it to the storage solution for long-term storage.

3) [Optional] If the cell contains organic anion transporters, Probenecid (1-2.5mM) or Sulfopyrazone (0.1-0.25mM) may need to be added to the cell culture medium to reduce leakage of the de esterification probe.

[Note (1)]: The storage solution of propiconazole or sulfamethoxazole is quite alkaline, so pH needs to be readjusted after adding it to the culture medium.

4) Add the prepared Fluo-8 and AM working solution to the cells, with the amount added to cover the cells. Incubate at 37 ℃ or room temperature for 20-60 minutes.

[Note (1)]: Regarding the incubation time, if the initial experiment cannot be determined, it is recommended to incubate for 30 minutes first to observe the fluorescence effect; If there is a significant amount of cell death, shorten the time appropriately; If the fluorescence intensity is too weak, extend the time appropriately.

【 Note (2) 】: Lowering the probe loading temperature may reduce the compartmentalization phenomenon of the probe.

5) Suck off the staining solution and replace it with HHBS or other physiological buffer (if necessary, use buffer containing transporter inhibitors such as 2.5mM propane sulfonic acid) to remove excess probes.

6) Use appropriate instruments such as laser confocal microscopy, flow cytometry, fluorescence enzyme-linked immunosorbent assay, etc., and detect with a wavelength of Ex/Im=490/520nm.

[Note (1)]: Fluo-8, AM enters the cell and is degraded by esterase to form Fluo-8, which is not retained in the cytoplasm in the form of valence binding. Therefore, it is not possible to fix live cells loaded with dyes and then detect Ca2+levels.

Precautions

1) The problem of uniform quenching of fluorescent dyes should be avoided from light as much as possible to slow down fluorescence quenching.

2) Acetoxymethyl ester (AM) is prone to moisture absorption. After taking it out of the refrigerator, please store it in a dry environment at room temperature before opening. Due to the trace amount of the reagent, please briefly centrifuge it before opening to ensure that the powder falls into the bottom of the tube.

3) Fluo-8 and AM will solidify and stick to the bottom, wall, or cover of centrifuge tubes at lower temperatures such as 4 ℃ and ice bath. They can be incubated at 20-25 ℃ for a moment until completely dissolved before use.

4) Fluo-8, AM is being used for the first time. It is recommended to prepare and use the storage solution immediately, divide it into single use quantities, and strictly seal and freeze it at ≤ -20 ℃ to prevent moisture. To ensure good experimental results, try to use it as quickly as possible.

5) For your safety and health, please wear lab coats and disposable gloves when operating.