Lasers & Fluorochromes

Choosing Fluorochromes

The choice of a fluorescent label is critical to the success of experiments in flow cytometry. Many commercially available antibodies are available that have been directly conjugated to highly purified fluorochromes. Acceptable fluorochromes for flow cytometry are:

• Fluorescein (FITC)
• Phycoerythrin (PE)
• PE-Texas Red (PE-TxRed) (NOT Texas Red)
• Peridinin Chlorophyll Protein (PerCP)
• Peridinin Chlorophyll Protein - Cy5.5 (PerCP-Cy5.5)
• Allophycocyanin (APC)
• PE-Cy7
• APC-Cy7

Dyes that require Violet laser-excitation include:

• Diamidino phenyl indole (DAPI)
• Hoechst
• Pacific Blue
• Pacific Orange
• Vybrant® DyeCycle™ Violet

Sytox Blue® and others see http://microbiology.utmb.edu/core/flowcore/scheduling/Vybrant%20DyeCycle%20Violet%20Stain.pdf.

Spectrum Viewer can be found here.

Reagents created using direct conjugation of fluorochromes to antibodies are generally superior to those requiring a labeled secondary antibody, since they require less washing steps during staining and produce lower background fluorescence.

APC	APC-Cy7
DsRed	eCFP
eGFP	FITC
PacificBlue	PE
PE-Cy7	PerCP
PerCP-Cy5.5	PE-TxRed
PI

These recommendations are from a White Paper published by Holden Maecker and Joseph Trotter at BD Biosciences…(http://microbiology.utmb.edu/core/flowcore/Schedule/Fluorochrome Selection.pdf):

• Pick the brightest set of fluorochromes available given your available instrumentation.
• If possible, avoid combinations of fluorochromes that have significant spectral overlap (see spectra in graphic above).
• Use the bar chart above to pick the brightest fluorochromes for dim antibodies, and the dimmest fluorchromes for bright antibodies.
• Spillover from bright cell populations into detectors requiring high sensitivity can cause loss of the ability to detect dim signals in the detectors deluged with massive fluorescent spillover.
• Since APC-Cy7 and PE-Cy7 can break down to APC and PE with harsh treatment, bad fixative, or with time - watch out for tandem dye degradation (particularly PE-Cy7 and APC-Cy7). This degradation can result in false PE and APC signals that are uncorrelated with real signals from antibodies intentionally conjugated to PE and APC and may be present in the sample dependent on experimental design.
• A stabilizing fixative is available that helps prevent degradation of PC-Cy7 and APC-Cy7 while still fixing cells (BD Biosciences Cat. No. 338036).
• It is worth noting that some cells are autofluorescent. In such cells it is often difficult to detect signals from bound antibodies, or other fluorescent probes, when the autofluorescence of the cells is the same color as the fluorescent tag in use. It is prudent to design experiments with this in mind and to select fluorochromes that do not emit fluorescence in the same spectral region as the cells in the experiment – typically in the green and yellow regions.
• For extremely autofluorescent cells, only PE-Cy7 and APC-Cy7 are likely to work since they emit in the near infrared and outside the autofluorescence region of most cells.