PE Fire 640

Excitation and Emission Spectra of PE/Fire™ 640

Emission spectra (top) and normalized emission spectra (middle) of PE/Fire™ 640 as run on a 5-laser Cytek® Aurora Spectral Cytometer. To compare PE/Fire™ 640 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

Normalized excitation and emission spectra (bottom) of PE/Fire™ 640 obtained from a spectrophotometer. To compare PE/Fire™ 640 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

Spectral Spillover of PE/Fire™ 640 

Spillover impact of PE/Fire™ 640 into detection channels of a 5-laser Cytek® Aurora Spectral Cytometer.

Multicolor Compatibility of PE/Fire™ 640 

Titration Curve for PE/Fire™ 640

PE/Fire™ 640 Stability

Photostability

Two conditions of anti-CD4 PE/Fire™ 640 photostability were tested: (1) antibodies formulated at the optimal test concentration were left under fluorescent lighting or protected from light, then used to stain human PBMCs (indicated as “Ab only” in the graphs) and (2) antibodies were used to stain human PBMCs, which were then fixed and left in FluoroFix™ either under fluorescent lighting or protected from light (indicated as “Ab+cells” in the graphs). The long-term stability of antibody conjugates can be predicted by its brightness (staining index, left) and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel (right).

Stability in Fixatives 

RBC-lysed human blood cells were stained with anti-CD4 PE/Fire™ 640 and fixed according to the recommended protocols for each fixative. Cells were analyzed directly after fixation and washing (fresh), or analyzed after being stored overnight in Cyto-Last™ Buffer. PE/Fire™ 640 is resistant to paraformaldehyde fixation, but is sensitive to organic solvent-containing phospho-specific fix and perm buffers (e.g. True-Phos™). The long-term stability of antibody conjugates can be predicted by its brightness (staining index, left) and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel (right). Learn more about our flow cytometry buffers.

Heat Stability 

Antibodies were aliquoted into a sealed vial to avoid evaporation and stored at 4°C, 22°C, or 37°C for up to 28 days. The long-term stability of antibody conjugates can be predicted by its brightness (staining index, left) and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel (right). PE/Fire™ 640 does not have a significant reduction in staining index over 28 days at room temperature (22°C), but is reduced by 40% in staining index after 28 days at 37°C. There is no change in compensation, and the loss of staining index is due to an increase in non-specific binding of the negative population, not a loss of fluorescence intensity.

Note: Percent compensation for PE/Fire™ 640 heat stability was obtained on a conventional flow cytometer. Percent compensation for each other experiment was obtained on a 5L Cytek® Aurora by measuring percent spillover from the tandem dye into the donor fluor (PerCP, PE, or APC) peak emission channel. Percent spillover and compensation values on a conventional cytometer may differ significantly.