AlphaLISA SureFire Biotin-Free Human and Mouse Total ATF-3 Detection Kit, 100 Assay Points

LPS induction of ATF-3 expression

RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with 1 µg/mL LPS for the indicated times at 37°C, 5% CO₂.

After treatment, the cells were spun down and lysed with 65 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ATF-3 levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 30,000 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, LPS treatment induced ATF-3 expression after 2 hours, reaching maximal levels after 6 hours.

R848 induction of ATF-3 expression

RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with 5 µM R848 at indicated times at 37°C, 5% CO₂.

After treatment, the cells were spun down and lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ATF-3 levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 40,000 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.

R848 is a TLR7/8 agonist that mimics pathogen-associated molecular patterns triggering innate immune responses. R848 induced ATF-3 expression after 2 hours, reaching maximal levels after 6 hours.

RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with increasing concentrations of LPS for 4 hours at 37°C, 5% CO₂.

After treatment, the cells were spun down and lysed with 65 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ATF-3 Total and IRF5 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 30,000 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, LPS treatment resulted in a dose dependent increase in the levels of ATF-3 with no significant changes in IFR5 Total signal.

RAW 264.7 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 LPS for 4 hours.

After treatment, the cells were lysed with 30 µL or 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ATF-3 and ERK Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 20,000 cells for ATF-3 Total and 6,000 cells for ERK 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, LPS resulted in a dose dependent increase in the levels of ATF-3 while ERK Total levels remained unchanged.

Assay specificity - knockout validation

ATF-3 levels were assessed in HCT-116 (WT) and ATF-3 KO (Abcam, ab266872) cell lines cultured to confluency in T175 flasks at 37°C, 5% CO₂. Cells were lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and ATF-3 levels were evaluated using the AlphaLISA SureFire Biotin Free 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.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. ATF-3 was detected in WT but not KO cells, confirming the specificity of the assay for the detection of ATF-3 protein.

ATF-3 expression in various cell lines

Adherent cells were grown to confluency in a T175 flask in complete medium and lysed with Lysis Buffer at a density of 2 x 10⁶ cells/mL. Suspension cells were harvested, spun down and lysed with Lysis Buffer at a density of 1.6 x 10⁶ cells/mL.

ATF-3 levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (20,000 adherent and 16,000 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.

ATF-3 is a stress inducible gene and basal expression was detected in a range of human cell lines and in RAW 264.7 mouse cells. As expected HCT-116 cells have elevated endogenous levels of ATF-3.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HCT-116 cells cultured to confluence in a T175 flask and lysed in 3 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and ATF-3 levels were evaluated using the AlphaLISA SureFire Biotin Free 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.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. This assay can detect endogenous ATF-3 expression in less than 2,500 HCT-116 cells.