BCECF, AM Fluorescence PH Assay Kit

 

Product Name	Cat.No.	Spec..
BCECF, AM Fluorescence pH Assay Kit	G1726-100T	100 T

 

 

Intracellular pH plays an important role in many cellular activities, such as cell growth, enzyme activity, ion transport, and other processes.BCECF, AM is a cell-permeable pH fluorescent dye that is widely used to monitor changes in intracellular pH by contrasting the ratio of dual excitation wavelengths. BCECF, AM is non-fluorescent and can be sheared into BCECF by intracellular esterases upon entry into the cell, thus being retained inside the cell.BCECF, AM has been widely used not only in mammalian cells, but also has been reported to be used for intracellular pH levels in animal tissues, plant cells, bacteria and yeasts.

This kit is based on the above detection principle, with BCECF, AM as the main body, and provides specialized matching buffer, which is easy to use and more sensitive to detect. It can be used to monitor the dynamic change of intracellular pH with fluorescence microscope, enzyme labeling instrument and other corresponding instruments.

 

 

Transported on dry ice; stored dry and protected from light at -20°C. Valid for 12 months.

 

 

Component Number	Component	G1726-100T
G1726-1	BCECF, AM fluorescent probe	20 μL
G1726-2	pH testing buffer	20 mL
Manual		1 pc

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

 

 

1. Pre-experimental preparations: Cells were inoculated in advance in 96-well plates at a certain density, and the cells were pre-treated accordingly according to the purpose of the experiment;

 

2. BCECF, AM assay working solution preparation: according to the need to prepare BCECF, AM assay working solution, the preparation 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 recommendation of the dilution ratio of 1:500;

 

	1 sample	10 samples	100 samples
BCECF, AM fluorescent probe	0.2 μL	2 μL	20 μL
pH testing buffer	100 μL	1 mL	10 mL

 

3. Staining markers (in the case of adherent cells; suspension cells need to be centrifuged and changed each time):

3.1 Remove the original cell culture medium and wash the cells 1-2 times with PBS or other buffers;

3.2 Remove the washing buffer, add 100 μL of BCECF, AM assay working solution to each well, and incubate for 30 min at 37°C in a cell culture incubator protected from light;

3.3 At the end of the incubation remove the BCECF, AM assay working solution and wash the cells 2-3 times using PBS or other buffers;

Note: Wash out the BCECF, AM assay working solution.

3.4 100 μL of buffer was added for fluorescence detection.

4. pH calibration (pH calibration by nigericin method as an example, optional):

4.1 Prepare buffers of different pH (e.g., 6.6, 7.0, 7.2, 7.4, 7.8, 8.2) in advance;

4.2 After staining and labeling, buffers of different pH as well as nigericin (final concentration of 10 μg/mL) were added respectively, and the fluorescence intensity was measured after incubation for 10 min at 37°C in a cell incubator protected from light;

4.3 Plotting of fluorescence intensity versus pH graphs for sample pH calculations.

Note: The nigericin method of calibrating pH is for reference only, you can choose the corresponding method according to your experimental needs. In general, the higher the fluorescence intensity ratio, the higher the pH.

5. Fluorescence assay: The intracellular pH level is detected and analyzed using an enzyme marker and other related instruments. Usually BCECF, AM is applied to the double excitation ratio: EX/EM=490/530 nm : EX/EM=440/530 nm.

 

 

1. Due to the different intercellular properties and pre-treatments, the proportion of probes used and the range of incubation times described in the steps are for reference only and can be adjusted on a case-by-case basis.

2. Fluorescent dyes are subject to quenching, so please try to avoid light to slow down the fluorescence burst.

3. BCECF, AM is very sensitive to humidity, when you use it for the first time, please divide it appropriately according to the experimental arrangement, and avoid repeated freezing and thawing.BCECF, AM assay working solution should be prepared and used now.

4. Shake well before use to ensure complete dissolution.

5. Based on the working principle of this probe, cells cannot be fixed or perforated.

6. For your health and safety, please wear a lab coat and gloves when operating.

 

For Research Use Only!