This technical note summarizes how protein detection works in the Cytos™ workflow on the Element Biosciences AVITI24™ system, using cyclic immunofluorescence, along with the validation data supporting it. It is a summary prepared for Velgen Technik; for the complete note, figures, and references, see the original Element Biosciences publication linked at the end.
Highlights
• Cytos Protein Kits use cyclic immunofluorescence to detect up to 20 protein targets per sample, at single-cell resolution, alongside cell morphology.
• The protein cartridge can be paired with a second cartridge for same-cell 3′ transcriptome and/or guide RNA sequencing.
• All 20 targets in the standard panels show localization and expression patterns consistent with published literature.
Introduction
The Cytos multiomics workflow on the AVITI24 captures cell morphology, protein expression, 3′ transcriptome, and guide RNA data in a single run. This note focuses on the protein workflow — its scope, chemistry, and the validation data that demonstrate specific, reproducible protein detection at single-cell and subcellular resolution.
Cytos Protein Workflow
The Protein Kit and its cartridge can be run standalone or combined with a second cartridge for same-cell transcriptome and guide RNA readouts. Standard panels focus on defined biological areas (4-plex focus-area panels), and custom probes are supported.
Approximate run times are about 9 hours for a 4-plex panel and about 22 hours for a 20-plex panel. The workflow includes a pause for protein data review before the RNA cartridge run, and outputs a combined per-cell Spatial Data Object that links protein signal to each segmented cell.
Protein Detection Chemistry
Detection uses biotinylated antibody probes carrying fluorescent labels, pooled as red/green 2-plex reagents for each imaging cycle, plus dye-based Cell Paint markers used for cell segmentation. Each cycle's signal is removed before the next antibody pair is applied, enabling many targets to be imaged on the same cells.
Results reflect relative rather than absolute protein quantity. Isotype antibodies serve as negative controls and TOMM20 as a positive control, with the appropriate comparison control chosen by fluorescence channel.
Concordance with Immunofluorescence Microscopy
Across the five panels, Cytos cyclic immunofluorescence showed qualitative side-by-side agreement with single-cycle reference immunofluorescence (IF) microscopy, confirming that the cyclic workflow reproduces expected staining patterns.
Specificity and Concordance with Literature
Examples from each panel — organelle markers, neuronal/glial markers, cytoskeletal/nuclear markers, immune subtyping, and cell signaling — showed the expected subcellular localization and cell-line specificity, consistent with published literature.
Detection Performance and Reproducibility
Detection uses an Otsu-based Intensity Above Threshold (IAT) metric, with an acceptance criterion of at least 2.0-fold signal over the negative control. For example, CD19 expression was detected in B-cell lines but not T-cell lines, demonstrating target specificity.
Run-to-run reproducibility was high, with R² above 0.9 for all panels tested. Signal stability was maintained across 10 imaging cycles, and channel crosstalk was assessed using a nuclear-ratio measure.
Summary
Cytos protein detection delivers IAT ratios above threshold, literature-consistent subcellular localization, high reproducibility (R² > 0.9), and concordance with reference IF microscopy — supporting integrated single-cell multiomics on the AVITI24, where protein data can be linked to morphology, transcriptome, and guide RNA from the same cell.
Source
This is a summary of the Element Biosciences technical note “Cytos™ Protein Detection on the AVITI24™ — Cyclic Immunofluorescence Detection for the Cytos Workflow.” All technical claims and data are from Element Biosciences. Read the full technical note, including figures and references, at elementbiosciences.com. To discuss Cytos for your lab, view the Cytos product page or contact our team.