AlphaLISA SureFire Biotin-Free Human and Mouse Total HPRT1 Detection Kit, 500 Assay Points

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with increasing concentrations of ARV-771 for 18 hours at 37°C, 5% CO₂.

After treatment, the cells were spun down and lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). HPRT1 and BRD4 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 10,000 cells) was 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. The plate was read on an Envision using standard AlphaLISA settings.

As expected, treatment with ARV-771 resulted in a dose-dependent decrease of BRD4 Total while HPRT1 Total levels remained unchanged.

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with increasing concentrations of palbociclib for 24 hours at 37°C, 5% CO₂.

After treatment, the cells were spun down and lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were further diluted and HPRT1 Total and Phospho (Thr821) Rb levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 5,000 cells) was 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. The plate was read on an Envision using standard AlphaLISA settings.

As expected, treatment with palbociclib resulted in a dose-dependent decrease of Phospho (Thr821) Rb while HPRT1 Total levels remained unchanged.

MOLT-4 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium and treated with increasing concentrations of palbociclib for 8 hours at 37°C, 5% CO₂.

After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were further diluted and HPRT1 and GSPT1 Total levels were evaluated using AlphaLISA SureFireassays. For the detection step, 10 µL of cell lysate (approximately 5,000 cells for GSPT1 Total or 1,000 cells for HPRT1 Total) was 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. The plate was read on an Envision using standard AlphaLISA settings.

As expected, treatment with CC-90009 resulted in a dose-dependent decrease of GSPT1 Total while HPRT1 Total levels remained unchanged.

HPRT1 Total expression in various cell lines

Adherent cells were plated at 40K cells/well and grown overnight in a 96-well plate in complete medium. Cells were lysed with 100 µL Lysis Buffer. Suspension cells were harvested and lysed with Lysis Buffer at a density of 3.2 x 10⁶ cells/mL.

HPRT1 Total levels were evaluated by AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (4,000 cells/datapoint) 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. The plate was read on an Envision using standard AlphaLISA settings.

HPRT1 expression was detected in a range of adherent and suspension cell lines.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HEK293 cells seeded in T175 flasks cultured to confluence and lysed in 16 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and HPRT1 levels were evaluated by AlphaLISA SureFire Biotin Free. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect HPRT1 expression in less than 100 cells.