Actome is using
the CYTENA F.SIGHT
to perform high-precision
single-cell dispensing
Actome is a biotech startup at the forefront of proteomics and interactomics research. It supplies clients with game-changing off-the-shelf Protein Interaction Coupling (PICO) technology and bioinformatics software for various molecular applications. Actome’s recent venture into single-cell proteomics solutions highlights its commitment to supporting its clients by developing the next generation of single-cell assays.
Industry
Biotech
CYTENA Instruments in Use
F.SIGHT
Using CYTENA Products Since
2023
Cell Types
Adherent Breast Carcinoma
Cell Line BT474 & Suspension
Histiocytic Lymphoma
Cell Line U937
Key Results
Background
Proteomics is crucial in modern drug discovery as most therapeutics target proteins to modify their expression, interactions, or post-translational modifications. Therefore, tools that can precisely evaluate the effects of potential therapies on proteins are more important than ever. The advent of single-cell omics has revolutionized our understanding of how therapies affect various cell types within complex tissues. By overcoming the limitations of bulk analyses, which homogenize thousands of cells and overlook individual variations, single-cell omics uncovers the inherent heterogeneity of cellular responses, providing deeper insights into drug activity. Advances in this area underscore the growing need for tools that combine single-cell seeding capabilities with assays capable of accurately and reliably measuring changes in protein abundance, interactions, and modifications.
Advanced proteomic techniques have massive potential for furthering our understanding of cellular signaling pathways and providing unparalleled insights into the effectiveness of various therapies. However, progress in this area faces several challenges, including the lack of instrumentation that maintains cellular viability while delivering efficient and accurate single-cell seeding. This problem becomes even more significant when dealing with extremely small liquid quantifies, such as picoliter-volume droplets. The highly competitive drug discovery landscape requires ongoing advancements in time and resource efficiency and solutions to address the challenges of complex sample preparation and high costs.
Challenge
Actome’s main challenge was combining high-throughput capabilities with high precision in single-cell proteomics workflows. Achieving this would enable them to offer their clients a single-cell PICO (scPICO) assay—a potential game changer for precisely analyzing protein expression, interactions, and modifications at the single-cell level.
Actome scientist, Dr. Christoph Niemöller noted:
“The main challenge was to deliver single cells to 12 nL
droplets under oil in 384 well plates with high precision.”
This was a considerable obstacle, as few instruments could provide the ultra-low-volume dispensing required for the scPICO assay. Alternative methods, such as FACS, were unsuitable because they risked compromising cellular viability and were incompatible with the required sample volumes.
Dr. Niemöller stated:
“We knew from experience that FACS sorting is not
precise enough to target the tiny droplets we are
working with.”
Solution
Actome's position as a leader in single-cell proteomics and its mission to provide cutting-edge technologies made the F.SIGHT from CYTENA an essential component of its laboratory infrastructure. It allowed the Actome team to tackle scPICO assay development with confidence.
The F.SIGHT’s dual imaging capabilities made it easy for Actome scientists to monitor single-cell dispensing into 384-well plates, instantly side-stepping issues associated with other methods and allowing them to place greater trust in their workflows.
Dr. Niemöller noted:
“Highly precise single-cell isolation to 384-well plates is key
for the success of our workflow. Additionally, the proof of
single-cell isolation is critical in order to control for empty
wells or doublet events.”
The F.SIGHT eliminated variables like empty and duplicate wells, a crucial advantage when establishing complex assays like scPICO. This allowed them to focus on optimizing other parameters without worrying about the precision and efficiency of single-cell dispensing.
Furthermore, the F.SIGHT preserved cell viability when handling picoliter volumes, addressing a perennial challenge in cell dispensing workflows.
Impact
The F.SIGHT facilitated the development of the scPICO assay, which showed incredible results in detecting the single-cell heterogeneity of phosphorylated 4EBP1 in human cell lines treated with dactolisib (a small-molecule inhibitor of PI3K and mTOR) (Fig. 1).
Figure 1. U937 cells were treated with dactolisib (5.6 μM) for 4 h or left untreated (DMSO) and subjected to the scPICO workflow using the F.SIGHT (left panel) or were treated with λ-phosphatase (PPase) and subjected to conventional bulk analysis without single cell isolation (right panel). Different proteoforms, including total and phosphorylated 4EBP1, were detected. Full application note.
The results generated using the F.SIGHT’s single-cell isolation capabilities accurately capture the heterogeneity between individual cells, an insight completely lost in the bulk analysis. Thus, the F.SIGHT allows researchers to gain a deeper understanding of individual cellular responses, enabling more accurate assessments of drug efficacy and mechanisms of action and increasing the translational impact of their research.
The development of the scPICO assay represents a significant achievement for Actome. Dr. Niemöller emphasized:
“The success rate of targeting the 12 nL droplet under oil is
something between 20 and 50 % depending on the cell
type and cartridge performance.”
Actome scientists also highlighted the straightforward maintenance of the F.SIGHT and its user-friendly cartridges, which significantly reduce the risk of contamination. Finally, they emphasized the F.SIGHT’s capacity to empower the next generation of single-cell assays.
When asked if he would recommend the F.SIGHT to others facing similar challenges, Dr. Niemöller had this to say:
“Definitely yes, most likely only the F.SIGHT is capable of
isolating single cells with enough precision.”
