Flow cytometry analysis with a blue laser
Blue light-excitable fluorophores play a critical role in standard multiparametric flow cytometry. With the growing availability of flow cytometers equipped with UV, violet, green, yellow, red and blue lasers, the blue laser continues to be an essential part of nearly every flow cytometry instrument, and additionally blue laser-excitable fluorophores are commonly used because of compatibility with Red Fluorescent Protein for live-cell analysis. The expansive Alexa Fluor dye and eFluor dye portfolios support multicolor immunophenotyping by flow cytometry and include a variety of dyes compatible with the 488 nm spectral line of the blue laser.
Step 1. Search for the marker of interest (e.g., “CD4” or “CD45”).
Step 2. Narrow results by conjugate, target species and other criteria using side filters.
The table below lists the properties of the most commonly used fluorophores for the blue laser.
|Fluorophore||Fluorophore type||Ex/Em (nm)||Relative intensity* (1 = lowest, 5 = highest)||Suggested bandpass filter (nm)||Uses|
|Alexa Fluor 488||Organic dye||490/519||3||530/30||A brighter alternative to FITC that has greater photostability, pH-independent fluorescence from pH 4 to 10, and almost the identical fluorescence spectra|
|Fluorescein (FITC)||Organic dye||490/525||3||530/ 30||Widely used fluorophore that is prone to photobleaching and exhibits pH-sensitive fluorescence between pH 5–8|
|PerCP||Protein||482/678||3||695/40||Peridinin chlorophyll protein complex (PerCP) is a protein with a large Stokes shift that is ideal for multiplex fluorescence detection|
|PerCP-Cyanine 5.5||Tandem||482/695||3||695/40||Tandem dye, resonance energy transfer from PerCP molecule to Cyanine 5.5 dye|
|PerCP-eFluor 710||Tandem||482/710||4||710/50||Tandem dye, resonance energy transfer from PerCP molecule to eFluor 710 dye; 2-3 fold brighter than PerCP-Cyanine 5.5 tandem|
|R-Phycoerythrin (R-PE, PE)||Protein||496, 565/575||5||575/26||Widely used fluorophore with a high quantum yield that is ideal for detection of low-density antigens|
|PE-Texas Red||Tandem||496, 565/615||3||590/40||Tandem dye, resonance energy transfer from PE molecule to Texas Red dye|
|PE-Alexa Fluor 610||Tandem||496, 565/628||3||590/40||Tandem dye, resonance energy transfer from PE molecule to Alexa Fluor 610 dye|
|PE-eFluor 610||Tandem||496,565/607||3||610/20||Tandem dye, resonance energy transfer from PE molecule to eFluor 610; alternative to PE-Texas Red dye|
|PE-Cyanine 5 (TRI-COLOR)||Tandem||496, 565/667||3||670/14||Tandem dye, resonance energy transfer from PE molecule to Cyanine 5 dye|
|PE-Cyanine 5.5||Tandem||496, 565/694||4||695/40||Tandem dye, resonance energy transfer Cyanine 5.5 dye|
|PE-Alexa Fluor 700||Tandem||496, 565/723||3||695/40||Tandem dye, resonance energy transfer from PE molecule to Alexa Fluor 700 dye|
|PE-Cyanine 7||Tandem||496, 565/767||4||780/60||Tandem dye, resonance energy transfer from PE molecule to Cyanine 7 dye|
|* The relative intensity of any fluorophore is dependent upon cell type being studied, reagent used, instrument and instrument configuration. The relative intensities provided are to be used as a guideline; observed intensities may vary.|
For Research Use Only. Not for use in diagnostic procedures.