The TREM-2 (Triggering Receptor Expressed on Myeloid Cells 2) family of receptors regulates the immune system's various cell types, such as neutrophils, monocyte/macrophages, microglia, and dendritic cells. TREM-2 requires the adaptor protein DAP12 for downstream signaling. Human cerebrospinal fluid (CSF) has been shown to include soluble TREM-2, which has been reported to be increased in people with multiple sclerosis and other inflammatory neurological illnesses compared to those without such conditions.
The AlphaLISA SureFire Human Total TREM2/DAP12 Complex is a sandwich immunoassay for the quantitative detection of total TREM2/DAP12 complex in cellular lysates, using Alpha technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Alpha SureFire kits can be used for:
Cellular kinase assays
Receptor activation studies
Screening
AlphaLISA SureFire Ultra TREM2/DAP12 complex assay principle
The AlphaLISA SureFire Ultra TREM2/DAP12 complex assay measures the TREM2/DAP12 complex level in a biological sample (e.g. cell lysate).
The AlphaLISA SureFire Ultra TREM2/DAP12 complex assay uses two antibodies which recognize TREM2 and DAP12 proteins, respectively. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the first assay antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture the second antibody, which is biotinylated. In the presence of the TREM2/DAP12 complex, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of TREM2/DAP12 complex present in the sample.
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 cell 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 allows for 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.
