Images of Connor's Work

  • Cor4U cells stained with MitoTracker dye and a nuclear stain

Connor Jacobson started working in the Harvard Program in Therapeutic Science in August 2015. He began his research working alongside Post-doc Sharon Wang of the Sorger Lab. Their research, sponsored by the FDA, involved exploring cardiotoxic effects seen in patients treated with tyrosine-kinase inhibitors (TKIs). TKIs have been seen to be effective in reducing cancerous symptoms as first- and second-pass therapeutics but have been noted to cause cardiotoxicity such as myocardial infarctions and abnormal heart beating patterns, among others. As a member of both the LSP and Sorger Labs, Connor’s primary duties include studying the longitudinal effects of a panoply of drugs dosed to cell types of interest (namely IPS-derived Cor4U cardiomyocytes) and investigating the ways in which these drugs effect the Cor4U as well as various cancer cell lines. By employing a pre-existing protocol of cyclic immunofluorescence (CycIF; developed by Dr. Jia-Ren Lin of the Sorger Lab), a plethora of directly conjugated and unconjugated antibodies have been validated by Connor in the Cor4U. CycIF is traditional, indirect immunofluorescence, however, once secondary antibodies have been added and the cells imaged, a fluorophore-inactivation solution (H2O2-based) is added to the cells, bleaching the fluorophores of the secondary antibodies. Following washes, the cells are re-stained with directly conjugated antibodies and re-imaged as subsequent “cycles.”

Beginning with Connor’s initial battery of optimization and troubleshooting experiments, a finely-tuned plate-based CycIF (p-CycIF) workflow was established and applied to the cardiomyocytes. By exploring how particular biomarkers clustered over time and dose at a single-cell resolution, Connor helped reveal which epitopes are indicative of cardiotox at the mechanistic and signaling-pathway level. The hope is to predict cardiotox when these TKIs are used in cancer patients and to offer combination therapies that may alleviate unwanted cardiotox.

Connor’s assay development tested over 160 directly conjugated and unconjugated antibodies that serve as the foundation for an open-source, real-time antibody validation database. As the most experienced in plate-based CycIF assays and as a cog in HiTS’ matrixed research group, Connor contributes to many projects allowing for the addition of a multitude of antibodies to the database. By tracking different experimental conditions amongst collaborators, this resource serves as a starting point for any CycIF assay. Since the genesis of this resource, Connor has expanded his antibody validation efforts to include 200+ antibodies in seven different cell lines. Antibody validation remains a continuous effort that a multitude of individuals contribute towards. Spearheaded by Connor, the goal is to create a simplistic database through which a researcher can precisely pinpoint which antibodies worked in particular cell types and drug conditions.

Following the application of p-CycIF to cardiomyocytes, Connor has been responsible for technical development and optimization of the p-CycIF workflow. Recently he has begun to further multiplex the high-dimensionality of the assay by incorporating commercially available reagents into the protocol, such as Thermo’s Zenon-labeling. By matching species and isotype, one is able to label unconjugated antibodies with whichever fluorophore is available for that particular species, allowing multiple antibodies of the same species to be tested in the same cycle as long as the isotypes and fluorophore of the antibody are distinct. Furthermore, Connor has optimized the incorporation of an Edu-label into p-CycIF through “click-chemistry” which allows the tracking of cells’ progression through the cell cycle. While EdU can only be used in the last cycle of CycIF, it produces a strong signal that makes gating cells in the G1, S, and G2 cell-cycle phases feasible. Connor has also optimized and incorporated MitoTracker dyes staining which serves as a vital stain to gauge cell health before CycIF experiments begin. With the addition of a Hoechst nuclear stain, it and MitoTracker can be effectively used to visualize cell integrity before subjecting cells to a biomarker panel of cyclic staining. Connor has also tested and quantified cell loss, bleaching intensity and signal decay of over 10 fluorophores, as well as which dilutions of antibodies work best in particular conditions/cell types.

More recently, Connor has been trained in tissue CycIF (t-CycIF), working with different tissue types provided by collaborating clinicians. Like with plate-CycIF, Connor has re-worked the way in which we perform tissue CycIF assays by creating a clean and concise protocol. For all CycIF assays, be it tissue or plate-based, researchers need to understand the best imaging conditions they can employ to extract the most meaningful information from their experiment. With the help of Connor's protocols and the image analysis team in HiTS, the pipeline through which we can multi-plex experiments has grown greatly since Connor has joined the CycIF research team.

Connor works tirelessly on establishing a standardized protocol for all cyclic immunofluorescence assays from initial experimental design to image registration and processing. By refining the current CycIF protocols down to the most minute details, Connor has streamlined the ways in which we perform high-throughput plate-based microscopy across multiple systems in HiTS. If you would like to perform a CycIF experiment or have specific questions as to how to best proceed, email him at:

With a degree in Neuroscience, Connor is interested in further exploring academic research, namely in the fields of immune-oncology and neuro-oncology in hopes of one day transitioning to the pharmaceutical industry.