AlphaLISA SureFire Ultra Human Total Ki-67 Detection Kit, 10,000 Assay Points

Total-AlphaLISA SureFire Ultra 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 Total-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

Total-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Total target protein 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 AlphaLISA SureFire Ultra quality.

MCF7 cells were seeded in a 96-well plate at 40,000 in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with increasing concentrations of palbociclib for 24 hours.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ki-67 Total and GAPDH Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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 Ki-67 levels, while GAPDH Total levels remained unchanged.

A549 cells were seeded in a 96-well plate at 20,000 cells/well in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with increasing concentrations of palbociclib for 24 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ki-67 Total and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of diluted cell lysate (approximately 800 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 Ki-67 levels, while ERK Total levels remained unchanged.

Decrease of Ki-67 levels in Dinaciclib treated cells

T-47D cells were seeded in a 96-well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with increasing concentrations of Dinaciclib for 24 hours.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ki-67 Total and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 cells for Ki-67 and 240 cells for Cofilin) 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, Dinaciclib triggered a dose-dependent decrease in Ki-67 levels, while Cofilin Total remained unchanged.

CDK6 PROTAC-mediated downregulation of Ki-67 expression in A549 cells

A549 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO₂. Cells were treated with 10 µM of CDK6 PROTAC, BSJ-03-123 for 24 hours.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ki-67, CDK6 and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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, degradation of CDK6 lead to Ki-67 downregulation in A549 cells while ERK Total levels remained unchanged.

CDK4/6 PROTAC-mediated downregulation of Ki-67 expression in A549 cells

A549 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 2.5 µM of CDK 4/6 PROTAC BSJ-03-204 for 24 hours.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Ki-67, CDK6 and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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, degradation of CDK4 and CDK6 lead to Ki-67 downregulation in A549 cells while no significant changes were observed in ERK Total levels.

Knockout validation of Ki-67 Total assay

Ki-67 Total protein levels were assessed in HeLa wild type (WT) and Ki-67 knockout (KO, Abcam, ab255407) cell lines cultured to confluency in T175 flasks. Each flask was lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking. Lysates were serially diluted in Lysis Buffer and evaluated for Ki-67 expression using the AlphaLISA SureFire Ultra assay. 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.

Ki-67 expression was only detected in WT cells, demonstrating assay specificity. Dotted line represents assay background.

Ki-67 expression in various cell lines

Adherent cells were grown to confluency in a T175 flask and lysed with Lysis Buffer at a density of 0.125 x 10⁶ cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer at a density of 0.4 x 10⁶ cells/mL.

Ki-67 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (1,250 adherent and 4,000 suspension 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. The plate was read on an Envision using standard AlphaLISA settings.

Ki-67 expression was detected in a wide range of human cell lines.

Ki-67 assay sensitivity - cell lysate

Cell lysate was prepared from A459 cells cultured to confluence in T175 flasks in complete medium. Cells were lysed in 8 mL of Lysis Buffer.

Lysate was serially diluted in Lysis Buffer and Ki-67 levels were assayed using the AlphaLISA SureFire Ultra kit. 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/datapoint is indicated. The dotted line represents assay background. The assay can detect Ki-67 expression in less than 200 cells.