Abstract
This protocol describes an In-Cell ELISA (ICE) methodology for the quantification of proteins and post-translational modifications in cells by immunocytochemistry with near infrared (IR) fluorescent dye-labeled detection. In this example, the cellular apoptosis marker cleaved PARP-1 is measured in Staurosporine treated human HeLa, HepG2 and HL-60 cells to determine half maximal effective concentration (EC50). The process involves seeding cells in a 96-well microplate where they are grown and treated. Following paraformaldehyde fixation and crosslinking to the plate, cells are then permeablized and blocked. Cleaved PARP-1 is detected using an In-Cell ELISA from the MitoSciences product range (Abcam-Eugene, USA) which contains an anti-cleaved PARP-1 mouse monoclonal antibody validated for specificity and sensitivity.
Quantification of cleaved PARP-1 is achieved by detection with IRDye-labeled secondary antibody and imaging achieved using LICOR Odyssey or Aerius system. Methods for both cultured adherent and suspension cells are also described in addition to a colorimetric detection method. In-Cell ELISA is advantageous over traditional Western blotting since this technique generates quantitative data, minimizes sample handling, allows high throughput analysis due to simultaneous treatments in a 96 well microplate, and allows a greater dynamic range and sensitivity.
Adherent cells can detach from a solid surface during apoptosis leading to an underestimation of the apoptotic cell proportion. The novel In-Cell ELISA methodology described here was specifically developed to eliminate the loss of apoptotic cells and therefore provides a method for analysis of both adherent and suspension cells. Additionally, the measured analyte signal can be normalized to cell amount determined by Janus Green whole cell staining, to further increase the assay precision.
In general, the rapid fixation of cells in situ, allows stabilization of in vivo protein levels and their post-translational modifications, eliminating the potential for error that could occur during preparation of protein extracts in Western blotting. The entire process is requires approximately 5 hours total assay time divided by an overnight incubation. Commercial kits include all materials, biologicals and reagents for sample analysis.
