Changes in intracellular and intracellular pH play an important role in physiological and pathological processes. Changes in intracellular pH can easily induce cell division, rapid growth and functional disorders. The reduction of extracellular pH to acidic is an important signal for cancer and other lesions. Therefore, the detection of pH in different areas inside and outside cells is helpful for the study of cell life processes and pathological mechanisms [1-3].

Traditional sensors are easily affected by non-pH factors such as sample concentration, complex samples, etc., and most of them cannot be accurately positioned. The pKa values ​​of recently reported pH-responsive fluorescent proteins almost exceed the physiological pH range, resulting in reduced sensitivity.

ANNA is an ideal ratiometric pH-sensitive probe. Its absorption and emission spectra change reversibly with changes in pH within the physiological pH range (pH 6-8) (as shown in Figure 1), and its absorption and emission spectra change in acidic and alkaline states. Both have high fluorescence quantum yields, and their pH response is not affected by coupling groups or materials (Figure 2) [4-5]. ANNA is widely used and has been successfully used to image fixed cells in different pH environments (Figure 3); to monitor the pH in living cells and the pH of the tumor microenvironment in vivo and in vitro.

Figure 1 Ratio-type pH fluorescent probe ANNA changes reversibly with changes in pH

Figure 2 The pH responsiveness of the ANNA probe is not affected by the coupling group or material [4]

Figure 3 ANNA was successfully used to image fixed cells in different pH environments [5]

J&K Scientific provides scientists around the world with the fluorescent probe ANNA transformed from the scientific research results of Associate Professor Liu Xiangjun:
Good response within physiological pH range;
High photostability and high fluorescence quantum yield;
The ionic strength does not interfere with fluorophores and can be used in complex biological systems such as living cells, tissues and animals;
Other groups can be easily coupled through the active ester method without affecting the pH response function.

ANNA, 95%, pH fluorescent probe
10 MG

Product instructions
Cells were placed in a 20 mm diameter glass-based cell culture dish (MatTek Co.) and cultured for 24 h;
pH probe ANNA (4 M) was incubated with cells in cell culture medium for 1-2 h;
Wash the cells three times with PBS buffer (pH=7.4) to remove free probe;
Cell imaging: observe with a 100x confocal microscope. The excitation wavelengths are 405nm and 488nm, and the fluorescence signal collects the fluorescence intensity of channel 1 at 425-475 nm and channel 2 at 500-600 nm [Note 1];
Make a pH standard curve: Treat cells with 1 mL of high potassium ion HEPES buffer [Note 3] containing 5 ug/mL nigericin [Note 2] at different pH for 10 minutes; perform cell imaging and draw it based on the relationship between I2 channel/I1 channel and pH. curve;
The ratio of the sample to be tested is brought into the standard curve to obtain the actual pH inside and outside the cell.
Note:
1: If equipped with a 455nm laser: it can be directly excited with 455nm to collect the fluorescence intensity of channel 1 (470nm-500nm) and channel 2 (505nm-540nm).
2: A carrier that carries potassium ions and hydrogen ions, which can quickly make the pH inside and outside the cell consistent.
3: High potassium ion HEPES buffer contains 125mM KCl, 20mM NaCl, 0.5mM CaCl2, 0.5mM MgCl2, 5mM glucose and 20mM HEPES.

References

1. Yan Wang, Nan Zhang, Shanshan Lu, Junyan Wang, Tao Bing, Xiangjun Liu, Chunyan Chen, Dihua Shangguan. Anal. Chem. 2019, 91, 13720-13728.
2. Jin Zhou, Canliang Fang, Tianjun Chang, Xiangjun Liu, Dihua Shangguan. J Mater Chem B, 1, 661-667 (2013).
3. Tiancong Ma, Yi Hou, Jianfeng Zeng, Chunyan Liu, Peisen Zhang, Lihong Jing, Dihua Shangguan, Mingyuan Gao. J Am Chem Soc (2017), 140(1): 211-218.
4. Xiangjun Liu, Linlin Wang, Tao Bing, Nan Zhang, Dihua Shangguan. ACS Appl. Bio Mater., 2019, 2, 1368–1375.
5. Yi Hou, Jin Zhou, Zhenyu Gao, Xiaoyu Sun, Chunyan Liu, Dihua Shangguan, Wensheng Yang, Mingyuan Gao. ACS nano, 9, 3199-3205 (2015).