APC/Fire™ 810
APC/Fire™ 810 expands the range of our spectral detection farther into the infrared than any of our previous fluorophores. On a 5-laser Cytek™ Aurora (UV/V/B/YG/R), APC/Fire™ 810 has very limited spectral spillover into any other channel except PE/Fire™ 810. This means that it can be assigned to any antigen with moderate to abundant expression, and can be used in combination with any other fluorophore. As such, it is very useful for identifying antigens expressed broadly in the cell sample. APC/Fire™ 810 is also very useful for detecting variably expressed markers, like markers of activation, since it emits so far outside of the autofluorescence range. These characteristics make APC/Fire™ 810 potentially very convenient to add into pre-existing panel designs with minimal adverse impact.
Excitation and Emission Spectra of APC/Fire™ 810
Emission spectra (top) and normalized emission spectra (middle) of APC/Fire™ 810 as run on a 5-laser Cytek® Aurora Spectral Cytometer. To compare APC/Fire™ 810 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.
Normalized excitation and emission spectra (bottom) of APC/Fire™ 810 obtained from a spectrophotometer. To compare APC/Fire™ 810 with other fluorophores, use our Fluorescence Spectra Analyzer tool.
Spectral Spillover of APC/Fire™ 810
Spillover impact of APC/Fire™ 810 into detection channels of a 5-laser Cytek® Aurora Spectral Cytometer.
Multicolor Compatibility of APC/Fire™ 810
APC/Fire™ 810 Unmixing
Panel 1:
Marker
|
Fluorophore
|
CD4
|
BV510™
|
CD3
|
Alexa Fluor® 700
|
CD14
|
Alexa Fluor® 647
|
CD19
|
APC/Fire™ 750
|
CD27
|
Spark NIR™ 685
|
CD56
|
PE/Cyanine7
|
Panel 2:
Marker
|
Fluorophore
|
CD4
|
APC/Fire™ 810
|
CD3
|
Alexa Fluor® 700
|
CD14
|
Alexa Fluor® 647
|
CD19
|
APC/Fire™ 750
|
CD27
|
Spark NIR™ 685
|
CD56
|
PE/Cyanine7
|
A common way to test the ability of a fluorophore to be unmixed from other fluorophores with heavily overlapping emission spectra is to create parallel staining panels where the fluorophore of interest (in this case, anti-CD4 APC/Fire™ 810) is replaced with a control antibody conjugated to a fluorophore with non-overlapping spectra (in this case, anti-CD4 BV510™). The accuracy of the unmixing controls, the pattern of the sample staining, and the percent positive populations are all indicators that APC/Fire™ 810 can be cleanly unmixed from all of the other fluorophores currently available for the red laser in this combination. Above are flow plots of RBC-lysed whole human blood cells stained with the two panels.
Intracellular Staining
PMA/Ionomycin-stimulated human PBMC were stained with either anti-CD3 BV421™ and anti-IFN-γ APC/Fire™ 810 (left), or with anti-CD3 BV421™ and anti-T-bet APC/Fire™ 810 (right). APC/Fire™ 810 is useful for both intracellular and intranuclear targets. Be aware that when constructing a multicolor panel, APC/Fire™ 810 might exhibit some sensitivity to solvents or oxidation conditions like photobleaching.
Titration Curve for APC/Fire™ 810
Titration curve of anti-CD4 APC/Fire™ 810 staining on human PBMC.
APC/Fire™ 810 Stability
Photostability
Two conditions of anti-CD4 APC/Fire™ 810 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 APC/Fire™ 810 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. 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 30 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). APC/Fire™ 810 has little reduction in staining index over 30 days at room temperature (22°C) and maintains 67% of the original staining index at 37°C after 30 days. The loss of staining index is due to an increase in non-specific binding of the negative population, not a loss of fluorescence intensity. There is a small change in compensation due to loss of acceptor fluors in the tandem.
Note: Percent compensation for each 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.
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