(115e) Multiplexed Live Cell Imaging Using Ultra-Fast Cycling

Live single cell protein analysis allows the study of dynamic cellular responses within heterogeneous cell populations such as tumors or differentiating stem cells. The ability to monitor live cells enables quantitative measurements of cellular responses and dynamic cell interactions. When these cells are profiled individually, we can better understand cell-to-cell differences and discover rare subpopulations that play essential roles in cancer metastasis, stem cell differentiation, and more. Protein abundance and its temporal and spatial distribution regulate cellular functions and therefore, quantification of proteins is critical for understanding molecular mechanisms of cellular processes.

The most widely used approaches for single cell protein quantification include genetically encoded fluorescent proteins, antibody-based methods, flow cytometry, mass cytometry (CyTOF), microfluidics, and mass spectrometry. These techniques have their own advantages but due to inherent tradeoffs, there is no single method that can meet the whole criteria of high multiplexing, high utility with clinical samples, and temporal and spatial mapping with time lapse measurements. For example, flow cytometry has highly multiplexed capability but it cannot provide spatial information of live cells. Live cell imaging provides temporal and spatial information but it does not allow high utility with clinical samples and high multiplexing.

To solve these limitations, we introduce a new live cell imaging technique that uses commercially available antibodies to profile protein expressions. We developed an ultrafast, gentle click chemistry method to quench and release fluorophores that allows multiplexed longitudinal measurements of heterogeneous live cells. We achieved highly efficient fluorescent cycling using a new generation of tetrazine (Tz)/trans-cyclooctene (TCO) probes at broadly nontoxic, nanomolar reagent concentrations (600 nM). After staining and imaging of live cells labeled with TCO-fluorophore conjugated antibodies, the fluorescence was quenched using Tz conjugated black hole quencher (BHQ3). The TCO-Tz click reaction triggers release of the fluorophores from the antibodies so that live cell imaging can be continued. (Fig. A) To test this reaction, we stained A431 cells with our probe conjugated anti-EGFR antibodies and quenched using Tz-BHQ3 where excellent staining and quenching were observed. (Fig. B). After 24 hours, we measured cell viability and any rebound signal. All cells were alive and proliferated during the time course and there was no observed rebound signal from previous staining.

To show the potential of using the probe for longitudinal live cell imaging, we chose a neutrophil differentiation model using immortalized hematopoietic progenitor cells. (Fig. C) Upon removal of β-estradiol, the progenitor cells differentiate into neutrophils over the course of 6-8 days. To keep track of the same non-adherent cells, we designed microwells using soft lithography and cultured cells inside the wells. (Fig. D) Using our probe, we imaged multiple markers for 6 days and observed differentiation into neutrophils of the same cells. (Fig. E) The cells were separatly measured using flow cytometry and marker expression from imaging matched well to that from flow cytometry. Here, we demonstrated the utility of our tool by monitoring and profiling differentiation over time. This opens up the opportunity to monitor and profile live cellular responses and states over the course of dynamic molecular changes.