Flow cytometry multicolor surface staining of human lymphocytes using anti-human CD4 (MEM-241) Pacific Blue™ antibody (4 µl reagent / 100 µl of peripheral whole blood) and intracellular staining of human lymphocytes using anti-human FoxP3 (3G3) Alexa Fluor® 488 antibody (prepared by MagicLink Alexa 488 NHS labeling kits, concentration in sample 3 µg/ml).
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BP Fluor 488 (Alexa Fluor 488 equivalent; AF 488) is a green fluorescent dye for the 488 nm argon laser line. BP Fluor 488 (Amax 493 nm, Emax 519 nm) has absorption and emission spectra comparable to Fluorescein (FITC), but are much brighter, far more photostable and less sensitive to pH changes between pH 4 and 10. BP Fluor 488 conjugates are compatible with common fluorescein equipment, settings, or filters and are ideally suitable for all applications in fluorescence microscopy and flow cytometry with high requirements in sensitivity. BP Fluor 488 shows more persistent and brighter fluorescence than Fluorescein in epifluorescence microscopes even without the addition of anti-fading agents to aqueous mounting media. In permanent organic mounting media BP Fluor 488 is also more fluorescent and long-term stable than FITC.
The features of BP Fluor 488 are the multiple negative charges which can significantly reduce conjugate aggregation in the bio application.
BP Fluor 488 can be conjugated to primary antibody, secondary antibody, or other biomolecules for the application below.
HaCaT cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes, permeabilized with 0.5% Triton X-100 for 3 minutes, and blocked with 5% FBS for 60 minutes. Then the cells were intracellularly stained overnight at 4°C with (A) 1: 100 diluted (5g/ml) Alexa Fluor® 488 rat IgG2a or with Alexa Fluor® 488 anti-human Cytokeratin 7 antibody (green) at (B) 1:100 (5g/μl) and (C) 1:200 2.5g/μl) dilution. Nuclei were counterstained with DAPI (blue). The image was captured with a 60X objective.