AlphaLISA SureFire Ultra Human binding TREM2 / DAP12 Detection Kit, 500 Assay Points

AlphaLISA SureFire Ultra TREM2/DAP12 complex two-plate assay protocol<

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well Optiplate plate before the addition of TREM2/DAP12 complex-AlphaLISA SureFire Ultra detection reagents. This protocol enables the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

AlphaLISA SureFire Ultra TREM2/DAP12 complex one-plate assay protocol

Detection of TREM2/DAP12 complex with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust AlphaLISA SureFire Ultra quality. 

TREM2 Activator rapidly induces TREM2/DAP12 Complex formation in THP-1 and primary macrophages

THP-1 cells were treated with 35 ng/mL TGFb  for 18 hours at 37°C, 5% CO2. Cells were harvested and seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA and then treated with 20 µM TREM2 Activator (MedChemExpress, Cat # HY-148095) for various time points.

After treatment, cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). TREM2/DAP12 Complex levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 16,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark.

TREM2 activator induced TREM2/DAP12 Complex formation within just 2 minutes, peaking after 10 minutes of treatment.

PBMCs were isolated from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (40,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO2. Cells were treated with 35 ng/mL TGFb for 18 hours and then treated with 20 µM TREM2 Activator (MedChemExpress, Cat # HY-148095) for various time points.

After treatment, cells were lysed in 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). TREM2/DAP12 Complex levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 8,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark.

As expected, TREM2 activator induced TREM2/DAP12 Complex formation very quickly, peaking after 10 minutes of treatment.

TREM2 Activator induces TREM2/DAP12 Complex in a dose-dependent manner

THP-1 cells were treated with 35 ng/mL TGFb  for 18 hours at 37°C, 5% CO2. Cells were harvested and seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA and then treated with the indicated concentrations of TREM2 Activator (MedChemExpress, Cat # HY-148095) for 10 minutes.

After treatment, cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). TREM2/DAP12 Complex, Total TREM2 and DAP12 levels were evaluated using respective AlphaLISA SureFire Ultra assays. Lysates were further diluted 1:10 in Lysis buffer for TREM2 and DAP12 Total assays. For the detection step, 10 µL of cell lysate (approximately 16,000 cells or 1,600 cells for Total TREM2 and DAP12) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark.

As expected, TREM2/DAP12 Complex formation was upregulated in a dose-dependent manner, while TREM2 and DAP12 total levels remained unchanged.