Application of common fluorescent dyes in flow cytometry



The development of flow cytometry has facilitated the development of more and more fluorescent dyes. Traditional fluorescent dyes such as FITC, PE, APC, percp, etc. are widely used in flow cytometry. In recent years, with the development of multi-color flow cytometry analysis technology, especially the analysis of more than six colors, some new fluorescent dyes have gradually been applied.

-Fluorescein with excitation wavelength of 488nm mainly includes FITC, PE, PerCP, PE-Cy5, PE-Cy5.5, PE-Texas Red, PE-Cy7, Alexa Flour 488
-Fluorescein with excitation wavelength of 633nm mainly includes APC, APC-Cy7, APC-H7
-Fluorescein with excitation wavelength of 405nm mainly includes Pacific Blue, BD Horizon V500, BD Horizon V450, Alexa Flour 405
-Fluorescein with excitation wavelength of 375nm mainly includes Hoechst 33342, DAPI

Also known as fluorescein isothiocyanate, it is a commonly used fluorescent dye, and its labeled antibody is suitable for all flow cytometers equipped with a 488nm argon ion laser. The maximum wavelength of FITC emitted after 488nm excitation light is 525nm , detected in the FL1 channel of the flow cytometer. It is worth noting that the fluorescence intensity of FITC is affected by pH value, and its fluorescence intensity decreases when the pH value decreases.

2. Alexa Flour 488
It is a green fluorescent dye whose emission is almost identical to that of FITC and can be detected by the FITC channel. The excitation maximum wavelength is 494 nm and the emission maximum wavelength is 519 nm, so Alexa Flour 488-labeled antibodies are suitable for use in all flow cytometers equipped with a 488 nm argon-ion laser. Moreover, the fluorescence is not easily quenched, and is suitable for a large pH range.

3. PE
Also known as phycoerythrin, the phase-labeled antibody is suitable for all flow cytometers equipped with a 488nm argon ion laser. The maximum wavelength of the emitted light after PE is excited by the 488 laser is 575nm, which is usually detected in the FL2 channel of the flow cytometer.

4. PerCP
Also known as polyalgal chlorophyll protein, when excited by a 488nm laser, the maximum wavelength of the emitted light is 677nm. Since PerCP, FITC and PE perform multi-color fluorescent staining, the overlap compensation of the fluorescence spectrum is very small, and the non-specific binding to myeloid cells is also less, so it is more used in related analysis. However, the quantum yield of PerCP is low, and it is best used in the detection of highly expressed antigens.

5. APC
Also known as allophycocyanin, its labeled antibody is suitable for all flow cytometers equipped with helium-neon laser or red laser laser, and its maximum emission wavelength is 660nm, which is usually detected by FL4 channel in BD flow cytometer.

6. PE-Cy5
It belongs to phycoerythrin conjugate. After the antibody labeled with Cy5 and the antibody labeled with PE are combined to form a complex, the energy can be effectively transferred from PE to Cy5. Because the overlapping spectral range of PE-Cy5 and PE is very small, PE-Cy5, PE and FITC can be used together in practical work, but it is not suitable to be used together with APC.

7. PE-Cy5.5
It is also a phycoerythrin conjugate. It is a fluorescent dye conjugated with PE and Cy5.5. Its maximum emission wavelength is 694nm. It can be used in combination with PE, FITC and APC. Easy to adjust.

8. PE-Cy7
A fluorescent dye composed of PE and Cy7 coupled with a maximum emission wavelength of 785nm, which can be used with PE, FITC and APC. However, it should be noted that PE-Cy7 has a strong fluorescence quenching property and needs to be strictly protected from light in the experiment.