Question
We have two CAR constructs against the same antigen that differ in their costimulatory domain. Before choosing one for a longer killing assay, we want a quick read of early activation: how many T cells turn on CD69 and CD25 after 24 hours with antigen-positive target cells?
Sources
June & Sadelain (2018) Chimeric Antigen Receptor Therapy. N Engl J Med 379, 64–73Review of CAR design, including the costimulatory domains used in clinical constructs.doi.org/10.1056/NEJMra1706169
Protocol
CAR T activation by flow · v1
- Thaw CAR-A and CAR-B T cells; rest overnight in medium with IL-2
- Co-culture with antigen-positive target cells at 1:1, 24 h, 37 °C, in a 96-well plate
- Controls: T cells without targets; PMA/ionomycin as positive control
- Stain live/dead, CD3, CD69, CD25; acquire on the flow cytometer
- Gate in FlowJo: lymphocytes → single cells → live → CD3+; export the workspace with the FCS files
Decided before the runReadout at 24 h only; 48 h moves to the killing assay notebook.
Plate layout
Results
Observation · A. FischerWell C6 (CAR-B) had under 2,000 live events and is left out, so CAR-B has n = 7.
ResultCAR-B shows more CD25+ cells than CAR-A after 24 h (median 55% vs 38%, n = 7 and 8 wells); CD69 differs less (72% vs 61%). The ranges don't overlap for CD25. The positive control is in range.
Conclusion
Early activation favours CAR-B in this example. Activation markers at 24 h don't predict killing or persistence, so both constructs go into the 72 h killing assay.
Provenance
SignedEach block carries its author's account signature.
Raw filesFCS files stay on the lab computer; the notebook stores gates, counts and plots.
page CAR-01 · 9 blocks · 2 authors · exported 22 Oct 2026