Multiplexed Fluorescence Plate Reader In Situ Protein Expression Assay in Apoptotic HepG2 Cells

Instead of Western blot being considered as a gold standard for intracellular protein expression assays, we developed a novel multiplexed high throughput (180 tests/day) in situ manual protein expression method directly in 96-well plates using 25,000-100,000 cells/well after formaldehyde fixation and Triton X 100 permeabilization. HepG2 cells were treated with ochratoxin A (OTA) and staurosporine (STP) to induce apoptosis. Antioxidant and apoptotic cell signaling protein expression were studied by various rabbit primary antibodies and HRP labeled secondary antibodies. The HRP labeled immune complexes were developed by H2O2/Ampliflu Red fluorogenic reagent and measured in a plate reader. Our assay can simultaneously quantify 22 protein antigens in one plate with 4 technical replicates with an interassay imprecision of <10% CV. The fluorescence signals are referred to total intracellular protein contents in the wells and given as fluorescence/protein ratio FPR, expressed as % of the controls (FPR %). OTA caused a dose-response increase (p < 0.05-p < 0.001) in SOD2, CAT, ALB, CASP3,7,9, BCL2, BAX, Nf-kB, phospho-Erk1/2/Erk1/2, phospho-Akt/Akt, phospho-p38/p38, and phospho-PPARg/PPARg levels while phospho-AMPK/AMPK ratios decreased (p < 0.05-p < 0.001). On the contrary, STP induced a dose-response decrease (p < 0.05-p < 0.001) in CASP3,7,9, BAX, BCL2, Nf-kB and phospho-Erk1/2/Erk1/2 expression while B-ACT, phospho-Akt/Akt, phospho-p38/p38 and phospho-PPARg/PPARg ratios increased.

Automated summary

We developed a novel multiplexed high throughput method for protein expression analysis that can directly analyze 25,000-100,000 cells in 96-well plates with a daily capacity of 180 tests. Our assay simultaneously quantifies 22 protein antigens with an interassay imprecision below 10% CV. The results show that ochratoxin A increased the expression of various proteins, while staurosporine decreased the expression of certain proteins.