Can I use fluorescent reporters in Cytos runs on the AVITI24™ system?
Fluorescent reporters are not recommended for use in Cytos runs. Fluorescent proteins, dyes, or sample autofluorescence can have significant impacts on the cell paint and protein performance of a Cytos run. The magnitude of the impact on protein or cell paint analysis can vary depending on the type of fluorescent protein used in the run.
Fluorescent reporters are used in cell selection and other custom assays. As the Cytos assay on AVITI24 uses fluorescent-based detection, using fluorescent reporters in Cytos runs impacts the resulting Cytos data. Internal testing of A549 cells with and without red fluorescent protein (RFP) demonstrated the greatest impacts on cell paint and immunofluorescence (IF)-protein metrics.
Figure 1. Impact of RFP on nuclear segmentation in A549 cells: (A) wild-type cells without RFP versus (B) cells with RFP. Nuclear stain (blue) defines the nucleus cell segmentation boundary (inner white lines) while other markers define the cell boundary. Nuclear segmentation fails or is inaccurate when the cell boundary and nuclear boundaries cannot be discriminated.
Endogenous fluorescent signal impacts cell paint and protein detection by increasing signal in certain channels, resulting in poor cell segmentation and boundary metrics. Figure 1 shows an example of the impact of RFP on nuclear segmentation.
Figure 2. Impact of RFP on nuclear localization of Ki67 in A549 cells: (A) wild-type cells without RFP versus (B) cells with RFP.
Signal from a fluorescent reporter in the run can impact localization and intensity measurements for protein targets, ultimately impacting the interpretation of protein data. For example, Ki67 typically binds to the nucleus and can be used to define the nuclear boundary. Figure 2 shows the loss of nuclear localization of Ki67 due to high background signal in A549 cells with RFP.
Endogenous fluorescent signal can also influence sequencing, with the most pronounced effects in the first batch when the signal is highest. Fluorescent signal could reduce overall alignment of raw 3’ transcriptome reads and targeted counts, but the impacts are generally minor. Fluorescent signal can also inflate levels of homopolymer reads in raw sequencing outputs if signal is high (homopolymer reads are filtered out during alignment). Fluorescent signal degrades over the course of sequencing due to laser exposure, so review the data across all batches and compare results to a non-fluorescent cell line to understand the impact on sequencing.
To minimize the impacts of fluorescent reporters on the Cytos assay, we recommend cloning out fluorescent reporters from your cell lines before performing a Cytos run. However, if you proceed with a Cytos run with a cell line expressing a fluorescent protein, the severity of the impact depends primarily on the type of fluorescent protein used in the experiment and the level of endogenous fluorescent signal.
All Cytos runs include a single background image at the start of the run before the cell paint and protein batches. There is also an option to save background images after each protein cycle. The mean cell and nuclear intensity are saved for background images into the RunStats and RawCellStats files, and batch background images are viewable in CytoCanvas™. Use these data to verify the presence or absence of background signal and check for changes to background signal over time.
The type of fluorescent protein and its spectral overlap with the AVITI24 system drive the severity of the impact on a Cytos run (i.e., more overlap results in more severe impact). The following table lists the excitation and emission ranges for AVITI24. Avoid overlap with these ranges:
|
Excitation (nm) |
Emission (nm) |
Potential Impact |
|
520 |
575–617 |
Sequencing |
|
536–570 |
Sequencing, Protein GRN |
|
|
638 |
685–748 |
Sequencing, Nucleus |
|
659–679 |
Sequencing, Protein RED |
Even outside these spectral ranges, all fluorescent proteins are likely to have some impact. Blue fluorescent protein (BFP) has the least overlap with the AVITI24 system and is therefore likely to have a less severe impact. Green fluorescent protein (GFP) also has less overlap, but still impacts Cytos results. Red and yellow fluorescent proteins (RFP, YFP) have the greatest overlap with the AVITI24 system and are likely to have a more severe impact on Cytos runs.
Mitigations such as photobleaching, quenching, or degradation are not recommended as these methods are likely to have downstream impacts on RNA integrity and sequencing. Internal testing of photobleaching methods showed increased background signal and lower overall RNA detection, though correlation with targeted Direct In Sample Sequencing (DISS) and 3’ transcriptome was stable between conditions.