Detection and performance assessment of FITC, AF488 and SF488

Compare detection windows, labeling chemistry, signal intensity, photobleaching and pH response separately.

Detection window and reactive form

FITC, Qihe AF488 and SF488 are candidates for green detection. FITC uses an isothiocyanate group, whereas NHS products use an activated ester. Check molecular weight, input and reaction conditions by catalog number. AF488 here denotes the Qihe product; parameters for original Alexa Fluor 488 require separate confirmation.

Signal intensity and photobleaching

Compare specific signal and negative-sample background at matched target concentration, similar labeling density and common instrument settings. Photostability requires repeated acquisition under fixed illumination and exposure; a single endpoint does not measure bleaching rate.

Reading the available flow example

In Qihe’s Mouse CD4 (GK1.5) example at 0.4 µg/test, CD4+ gate fractions are 23.08% for SF488 and 20.59% for Qihe AF488. These are event fractions, not fluorescence intensity. The comparator is Qihe AF488; the result does not rank brightness or photostability against original Alexa Fluor 488. See the original SF example on the homepage.

Assess the intended environment

For acidic environments or time-lapse imaging, use product-specific pH-response and photobleaching data. A dye change also requires assessment of recovery, specific binding, negative background and storage stability.

References

Thermo Fisher · Detection-channel reference

documents.thermofisher.com

Fluorescent-labeled antibodies: Balancing functionality and degree of labeling

Vira S, Mekhedov E, Humphrey G, Blank PS. · Analytical Biochemistry · 402(2):146–150 · 2010

DOI: 10.1016/j.ab.2010.03.036

View the original SF flow-cytometry example →

Related AF products

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