If desired, stain with Janus Green and measure relative cell seeding density in an IR imager or microplate spectrophotometer.
Whole cell staining with Janus Green (optional)
Add an equal volume (100 µL) of 8% paraformaldehyde solution to fix and cross-link the cells to the microplate.Treat cells as desired in total volume of 100 µL media for 96-well microplate (or ¼ of volume of 384-well microplate).Seed cells into 96-well microplate at desired density (for 384-well microplate, seed at ¼ of the density).Reagent preparation and hints and tips for a successful assay are included in the appendices. Below, we provide a general protocol for a 96-well microplate that can be used for all our In-Cell ELISA kits as well as with our antibodies characterized for In-Cell ELISA. Kits include highly-specific, well-characterized primary antibodies generated from mouse or rabbit, and appropriately conjugated secondary antibodies for colorimetric, fluorescent or infra-red detection. Dual targets can be quantified using IR-conjugated secondary antibodies in our In-Cell ELISA kits. Detection can be colorimetric or fluorescent for a single target using our In-Cell ELISA kits or primary antibodies characterized for In-Cell ELISA. After fixation and permeabilization, primary antibody is added to the well and is incubated, followed by addition of a labeled secondary antibody. Cells are cultured (and treated if required) and seeded into a coated 96-well microplate. All of these data can have an automated export to Excel and PDF for example.In-Cell ELISA (also known as cell based ELISA, in cell western or cytoblot) is an immunocytochemistry method used to quantify target protein or post-translational modifications of the target protein, in cultured cells.
The data measurements can also be automated, substraction with reference wavelenght, cut off value for direct visualization of your data. Process 10, 30 or 50 microplates automatically and efficiently, freeing your time for other tasks. To automate reading processes for multiple plates, a robot-friend configuration of the plate readers interfaces with BioStack Microplate Stacker. The microplate readers are automation compatible to create walkaway processing. This comes with Gen5 software for additional powerful data analysis. Moreover, the microplate readers are suitable for performing UV-Vis absorbance measurements. Our microplate washer is designed to control the procedure of washing experimental samples arranged in plate-based formats and can be used to wash away background signals related to unbound, conjugated antibodies. At BioSPX, we offer many solutions that complete the ELISA workflow through high-quality equipment with one goal: An optimized workflow.