CAR T cell and B cell co-cultures – identifying phenotypes in co-culture


We want to observe CAR T cells killing B cells and count how many B cells one T cell can kill.

Consider this phase-contrast image sequence of CAR T cells killing malignant B cells in co-culture:



The first task is to identify the phonotype of each cell. Because B cells are only slightly larger than T cells ( B cells are about 10-12 um and T cells are about 7-8 um) it becomes difficult to use morphology alone. So in this case, we need a label on at least one of the cells. 

Here we demonstrate B cells labeled with tdTomato fluorescent protein.


The Red fluorescing marker deactivates upon apoptosis, thereby providing a cell death indication in addition to allowing us to discriminate T cells from target B cells.  In the movie directly above, you can see that over time the B cells disappear- this is the deactivation due to apoptosis induced by the T cells.

When we train our ML models on the combined input of the phase contrast images and the fluorescence images, we are able to classify each cell by phonotype. In the next movie, the T cells are marked with blue ‘+’ signs, while the living B cells are marked with green ‘+’ and the dead B cells are marked with a red ‘+’.



tdTomato is an exceptionally bright red fluorescent protein—6X brighter than EGFP. tdTomato’s emission wavelength (581 nm) and brightness make it ideal for live animal imaging studies. The tdTomato fluorescent protein is as photostable as mCherry (Shaner et al. 2004). Sequence mutations were introduced into a monomeric variant of DsRed through directed evolution by the Tsien lab in order to produce tdTomato. This very bright red fluorescent protein was made even brighter by creating a tandem dimer version, the tdTomato fluorescent protein. Because tdTomato forms an intramolecular dimer, it is considered monomeric (Shaner et al. 2004).

For more information on tdTomoto click here for the vendor website.