HTRF Human Total ITK Detection Kit, 10,000 Assay Points

Total ITK two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of total ITK HTRF detection reagents. This protocol allows the cells viability and confluence to be monitored.

Total ITK one-plate assay protocol

Detection of total ITK with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol allows miniaturization while maintaining robust HTRF quality.

Validation of HTRF total ITK assay for monitoring PROTAC-induced target degradation

Jurkat cells were seeded in a 96-well tissue culture-treated plate (200,000 cells/well) in complete culture medium and incubated for 24 hours prior to the experiment, at 37°C in a humidified atmosphere containing 5% CO₂. Cells were then treated for 4 hours with increasing concentrations of BSJ-05-037 or TL12-186, either alone or in the presence of the proteasome inhibitor epoxomicin (1 µM) or the neddylation inhibitor MLN4924 (3 µM).

BSJ-05-037 is a selective ITK-targeting PROTAC, whereas TL12-186 is a cereblon-dependent multi-kinase degrader.

Following treatment, cells were lysed with supplemented Lysis Buffer #4 for 30 minutes at room temperature under gentle shaking. For detection, 16 µL of cell lysate were transferred into a 384-well low-volume white microplate, and 4 µL of HTRF total ITK detection reagents were added. The HTRF signal was measured on a multimode microplate reader following an overnight incubation at room temperature.

In parallel, cell viability was assessed using the ATPlite™ 1step Kit (Revvity, #6016736/1/9). Five microliters of the same cell lysate were transferred into an ATPlite-compatible 96-well low-volume white microplate, and 25 µL of ATPlite 1step detection reagent were added. Luminescence was measured using an EnVision™ Nexus reader after a 10-minute incubation.

Both PROTACs induced a concentration-dependent reduction in total ITK levels in Jurkat cells after 4 hours of treatment, as measured with the HTRF Total ITK assay, without affecting cell viability. ITK degradation was rescued by co-treatment with either epoxomicin or MLN4924, confirming that the observed effects were dependent on proteasome-mediated degradation and the activity of the Cullin-RING E3 ubiquitin ligase complex.

Selective detection of endogenous ITK in Jurkat (ITK+) and MV4-11 (ITK-) cells

Jurkat cells, a human T-lymphocyte cell model, are known to express ITK, whereas MV4-11 cells, a human leukemia cell model, are not expected to express this kinase. Consistent with this observation, publicly available transcriptomic datasets indicate that ITK mRNA expression is undetectable or only present at negligible levels in MV4-11 cells.

Both cell lines were seeded in a 96-well tissue culture-treated plate (400,000 cells/well in 30 µL of complete culture medium) and incubated overnight at 37°C in a humidified atmosphere containing 5% CO₂.

Following incubation, cells were lysed with 10 µL of supplemented Lysis Buffer #4 (4X) for 30 minutes at room temperature under gentle shaking. For detection, 16 µL of cell lysate were transferred into a 384-well low-volume white microplate, and 4 µL of HTRF Total ITK detection reagents were added. The HTRF signal was measured on a multimode microplate reader following an overnight incubation at room temperature.

As expected, a robust HTRF signal was detected in ITK-expressing Jurkat cells, whereas no signal above background was observed in MV4-11 cells. These results demonstrate the ability of the HTRF Total ITK assay to specifically detect endogenous ITK expression in cellular lysates.