Fluo-4 AM Fluorescent Calcium Ion Detection Test Kit, 100 T

Product Information

Product Description/Introduction

Fluo-4 is an upgraded version of Fluo-3 commonly used in biological calcium ion detection experiments, the main difference is that the two chlorine atoms on the Fluo-3AM molecule are replaced by fluorine on Fluo-4AM, and the small change in the structure can make Fluo-4AM load faster and the detection signal is stronger at the same concentration. Because the AM group of Fluo-4AM probe not only covers the molecular part of Fluo-4 chelated calcium but also enhances its hydrophobicity, it can easily penetrate the living cell membrane and enter the cell. When Fluo-4AM enters the cell, it will be clipped into Fluo-4 by lactase, thus remaining in the cell. Fluo-4 itself has little fluorescence, but when combined with intracellular calcium ions, it can produce strong fluorescence. The fluorescence intensity is proportional to the intracellular calcium content.

This kit is mainly based on the above detection principles, Fluo-4AM as the main body, and provides a special matching buffer, easy to use, more sensitive detection. The content of calcium ions in living cells or the dynamic change of calcium ions in cells can be measured by fluorescence microscope, flow analyzer, enzyme labeler, etc.

Storage and Shipping Conditions

Ship with dry ice; Store at -20 away from light, valid for 12 months.

Product Content

Note: The above kit reaction times correspond to the 96-well plate assay system.

Assay Protocol / Procedures

1. Pre-experiment preparation: Cells are inoculated in 96-well plates and pre-treated accordingly to the experimental purpose;

2. Configuration of Fluo-4 AM working solution: Prepare Fluo-4 AM working solution according to the need, the configuration system can be referred to the following table, note that different cell characteristics are different, according to the actual situation of the experiment can be added or reduced the concentration of the probe used, the initial recommended dilution ratio of 1:500;

3. Staining markers (with adherent cells as an example; suspension cell need fluid change every time, so please perform centrifugation by yourself):

3.1. Remove the original cell culture medium and wash the cells 2-3 times with PBS or other buffers;

3.2. Remove the washing buffer, add 100μL Fluo-4 AM working solution to each well, and incubate in a cell incubator at 37 for 30min away from light;

3.3. Remove the Fluo-4 AM working solution at the end of the incubation and wash the cells 2-3 times using PBS or other buffers;

3.4. Replace with calcium ion detection buffer and continue incubation for 15-30min to ensure adequate hydrolysis of Fluo-4AM in cells (optional);

4. Fluorescence detection: Use fluorescence microscope, enzyme marker and other instruments to carry out its detection. Fluo-4 AM is green fluorescent with EX/EM 494/528 nm.

Note

1. Due to different intercellular properties and different pre-treatment methods, the ratio of probe use and incubation time range described in the steps are for reference only and can be adjusted according to specific circumstances.

2. Fluorescent dyes have quenching problems, please pay attention to avoid the light operation to slow down the fluorescence quenching.

3. Fluo-4AM is very sensitive to humidity, so please pack it properly according to the experimental arrangement when using it for the first time. The Fluo-4 AM Assay Working Solution should be prepared ready to use.

4. Some precipitation after thawing is a normal phenomenon, and it is fully shaken before use to ensure that it is completely dissolved.

5. Based on the working principle of this probe, it is not possible to treat the cells such as fixation or perforation.

6. For your health and safety, please wear lab coat and gloves during operation.

For Research Use Only!

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