Nexcelom Bioscience

978-327-5340

Directly Measure CAR T Cell-mediated Cytotoxicity using Direct Cell Counting in 96-well Plates

  1. Scan the same plate over multiple time points to directly monitor the killing of target cells.
  2. Perform whole well imaging and analysis of an entire 96-well plate in under 15 minutes
  3. No need trypsinize the cells and then perform lengthy flow cytometry protocols. The Celigo directly counts and reports every remaining target cell in each well

Introduction to CAR T Cell-Mediated Cytotoxicity

The Chimeric antigen receptor (CAR-T) technology focuses on generating a genetically modified t-cells that can directly bind to, and attack cells of interest. The CAR-T cell is comprised of an antigen-specific binding domain that is fused to the genetically engineered activating motif. Once expressed on the cell surface the CAR-T-cell can then target and attack the specific targeted cells.

Protocol

  1. Target cells (either stably expressing GFP or labeled with a fluorescent dye) are seeded in the wells of microplates
  2. CAR-T cells were added to the wells at different Effector:Target (E:T) cell ratios
  3. Whole well images are acquired for each channel and the images are automatically analyzed to report the number of remaining GFP positive target cells

In-plate Live-cell Imaging and Quantification of CAR T Cell-Mediated Cytotoxicity

  1. GFP-HEK293 cells expressing HIV viral envelop protein were seeded in 96-well plate
  2. CAR-T cells were then added to each well at different effector : target (E:T) ratios
  3. Subsequently the plate was scanned at 0, 18, 22 and 42 hours post addition of effector cells

Fluorescent and counted images of target cells at different E:T ratios

Live-cell Imaging of CAR-T Cell Cytotoxicity

The raw images on top show the GFP-HEK293 cells at different E:T rations. The greatest number of surviving GFP-HEK293 cells are those that contained the fewest number of CAR-T effector cells. The bottom set of images shows GFP-HEK293 cells that were identified and outlined by the Celigo software

  1. Since the plate was first imaged at 0 hours, we were able to establish a baseline for the number of GFP-HEK293 positive cells in each well
  2. All data was then normalized to time 0 hours

CAR-T cell induced cytotoxicity / Untransduced PBMC

Quantification of CAR-T Cell Cytotoxicity

The y-axis is showing the fold change in the number of GFP positive HEK293 cells in comparison to 0 hour time point. The greatest CAR-T cell killing was observed after a 42 hour incubation period. The CAR-T cells were effective in killing the HIV expressing HEK293 cells at both 1:1 and 1:2 E:T ratio

Celigo software shows and reports plate-level results: (42 hr time point)

Celigo plate-level results for CAR-T Cell Cytotoxicity

The entire plate was scanned and analyzed in 10 minutes. The plate level view shows a thumbnail picture of the full well scan as well as the number of GFP positive cells the software identified and counted.

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