Fluorescein is divided into four categories according to the excitation wavelength, which are mainly 375nm, 405nm, 488nm and 633nm. Among them, fluoresceins with excitation wavelengths of 488 nm and 633 nm, such as FITC, PE, APC, PI, etc., are commonly used.
Fluorescein with excitation wavelength of 488nm
1. Fluorescein isothiocyanate
Fluorescein isothiocyanate, FITC is the most widely used fluorescein, it emits bright yellow-green fluorescence after laser excitation, and the maximum emission wavelength is 525nm. The fluorescence intensity of FITC is greatly affected by pH, and often weakens with the decrease of pH. Therefore, special attention should be paid to the pH of the solution when using FITC.
2. Phycoerythrin
Phycoerythrin, PE is a common dye isolated and purified from red algae. It emits orange-yellow fluorescence after laser excitation, and the maximum emission wavelength is 575nm. PE has the characteristics of good light absorption performance and high photon quantum yield, and can be used to label proteins with low expression levels. In flow cytometry assays, PE-labeled antibodies are available on all flow cytometers equipped with a 488 nm argon-ion laser.
3. Alexa Fluor 488
Alexa Fluor is a series of fluorescent dyes developed by American Molecular Probes. The emission spectrum of Alexa Fluor 488 is almost the same as that of FITC, it emits green fluorescence after excitation by laser, and the maximum emission wavelength is 519 nm, but its fluorescence intensity and stability are better than FITC. In general, Alexa Fluor 488 maintains good photostability in the pH range of 4-10.
4. PE-Cy5
PE-Cy5 is a complex dye belonging to phycoerythrin conjugates. In this complex dye, the excitation energy can be transferred from PE to Cy5, and the maximum emission wavelength is 667nm. Because PE-Cy5 and FITC and PE have a small spectral overlap and less fluorescence interference, PE-Cy5 is often used in combination with FITC and PE in experiments. It should be noted that PE-Cy5 is not suitable for use with APC, the spectral overlap of the two is large.
5. PE-Cy5.5
Composite dye, energy is transferred from PE to Cy5.5, and the maximum emission wavelength is 694nm. Unlike PE-Cy5, the fluorescence interference between PE-Cy5.5 and APC is small and can be used together. Moreover, the fluorescence intensity of PE-Cy5.5 was better than that of PE-Cy5.
6. PE-Cy7
Composite dye, the maximum emission wavelength is 785nm. PE-Cy7 has no spectral overlap with FITC and less overlap with APC, so it can be used with FITC, PE, and APC. It should be noted in the experiment that PE-Cy7 has a strong light quenching property and needs to be absolutely protected from light.
7. PerCP-Cy5.5
Composite dye, the maximum emission wavelength is 695nm. PerCP has a low photon yield and is suitable for the detection of proteins with higher expression levels, while PerCP-Cy5.5 has a high photon yield and can be used for the detection of proteins with lower expression levels. When testing with APC on a dual laser tube instrument, compensation adjustment is required, but it is less than PerCP compensation.
8. PI/7-AAD
The maximum emission wavelengths of propidium iodide (PI) and 7-AAD (7-amino-actinomycin D) are 617 nm and 647 nm, respectively, and are commonly used for apoptosis detection. On most flow cytometers, PI is detected in the FL2 or FL3 channel, and 7-AAD is detected in the FL3 channel. 7-AAD has similar fluorescence characteristics to PI, but its emission spectrum is narrower than that of PI, and it has less interference to other detection channels. It is the best substitute for PI in multicolor fluorescence analysis.
Fluorescein with excitation wavelength of 633 nm
1. Allophycocyanin
Allophycocyanin, APC has a maximum emission wavelength of 660nm, and its labeled antibody is suitable for all flow cytometers equipped with helium-neon lasers. The detection channel is generally the FL4 channel. FITC, PE and APC are a classic 3-color combination.
2. Cy5
Cy5 is a small molecule dye with a maximum emission wavelength of 670nm. The labeled antibody is suitable for all flow cytometers equipped with a 633nm argon ion laser. The detection channel is generally the FL4 channel. In addition to flow cytometry, Cy5 is equally applicable to conventional fluorescence microscopy techniques. It should be noted that Cy5 has many non-specific bindings to monocytes and granulocytes, and the experimental results are prone to false positives.
3. Alexa Fluor 647
Alexa Fluor 647 is a bright red fluorescent dye with a maximum emission wavelength of 668 nm, typically detected in the FL4 channel of a flow cytometer. Alexa Fluor 647 has the characteristics of high photon quantum yield, good photostability, wide adaptability to pH range and not easy to be quenched. It is an excellent substitute for APC and Cy5.
4. APDye Fluor 647
APDye Fluor 647 (Alexa Fluor 647 equivalent; AF647) is a bright green-fluorescent dye optimal for use with the 633, 650 nm Argon laser. APDye Fluor 647 is structurally similar to Alexa Fluor 647, and spectrally is almost identical to Cy5 Dye, Alexa Fluor 647, CF 647 Dye, or any other Cyanine5 based fluorescent dyes. The dye is water soluble and pH-insensitive from pH 4 to pH 10. The dye has 4 sulfonate groups make it high water soluble and less aggregation in the aqueous solution . APDye Fluor 647 647 is used for protein and antibody labeling, or nucleic acid applications with high labeling density.
Fluorescein selection and matching
Various fluorescein-conjugated antibodies are essential in cell immunological detection and immunophenotyping experiments by flow cytometry. The selection and matching of fluorescein are usually considered from the following five aspects:
1. Configuration of the flow cytometer
The instrument needs to be equipped with a laser tube that can excite the corresponding fluorescein; for multi-fluorescein experiments, the instrument can simultaneously detect the fluorescence of multiple cells.
2. The brightness of the dye is adapted to the amount of antigen expression
For antigens with low expression, high-brightness fluorescein is recommended; for antigens with high expression, low-brightness fluorescein is recommended.
3. Minimize spectral overlap between fluoresceins
Try to choose a fluorescein combination with a small spectral overlap, such as FITC/PE-Cy7; choose a fluorescein combination excited by different lasers, such as FITC/APC, PE/APC.
Simultaneous detection of fluoresceins with severe spectral overlap will lead to fluorescence leakage and interfere with the detection results, so adjustment and compensation are required.
4. Avoid false positives caused by the combined use of complex dyes
Common composite dyes include PE-Cy5, PE-Cy5.5, PE-Cy7, PerCP-Cy5.5, APC-Cy7, etc. The signal decay of composite dyes will reduce the sensitivity of APC or PE. If signal decay occurs, then:
● reduce exposure of samples to light, heat or fixatives;
● Consideration when choosing dyes: The signal decay of complex dyes will reduce the sensitivity of APC or PE, and avoid the labeling of dyes and derivatives on the same cell. Choose the more stable tandem-dye (PerCp-Cy5.5);
● If the sample needs to be fixed, choose a stable fixative paraformaldehyde that can effectively prevent the decline of the complex dye signal: fix on ice for 10 minutes, remove the paraformaldehyde by centrifugation, and resuspend the sample in PBS.
5. Try to use red laser to excite samples with high autofluorescence
Each cell population has different levels of autofluorescence. Autofluorescence was observed in all fluorescence channels, but the autofluorescence intensity decreased rapidly at longer wavelengths. For cells with strong autofluorescence, choose red light for excitation, and fluorescein with a long emission wavelength (such as APC) can obtain a better S/N ratio; There is no obvious improvement in the increase of N, and FITC can be used.
Flow cytometry has high requirements on the selection and matching of fluorescein, especially for multicolor experiments, the effect of fluorescein matching directly affects the detection effect.
