The microplate can now be stored at 4°C for several days. For prolonged storage, supplement PBS with 0.02% sodium azide. Cover the microplate with the provided seal. Gently aspirate the paraformaldehyde solution from the microplate and wash the microplate 3 times with 300 μL 1X PBS per well.Immediately add an equal volume (100μL) of 8% paraformaldehyde solution to the wells containing culture media.When treating cells with drug of interest, we recommend including wells with untreated cells and cells treated with the drug solvent. (Duration of cell treatment should be determined by the user). Treat the attached cells as desired in 100 μL media (up to 10% serum is acceptable).Adherent cells can be grown in the recommended assay microplates or seeded directly into the assay microplate and allowed to attach for several hours or overnight. The optimal cell seeding density is cell type dependent and should be determined for each experiment ( see Appendix 1). For example, HeLa cells should be seeded between 25,000 - 50,000 cells per well for a 96-well microplate.For IRDye ® use a LI-COR ® Odyssey ® or Aerius ® near-infrared imaging system. Appropriate microplate reader: For HRP detection, use a microplate reader capable of measuring absorbance at 650 nm (preferably in a kinetic mode) or 450 nm.Clear bottom (black wall necessary for IRDye ® only), preferably coated (amine/collagen) for optimal cell culture. 0.3% solution Janus Green Stain (ab111622) in water.10X Phosphate Buffered Saline (PBS) (80 mM Na 2HPO 4, 14 mM KH 2PO 4, 1.4 M NaCl, 27 mM KCl, adjust pH to 7.4 with NaOH).For HRP detection, HRP substrate solution.Appropriate IRDye ® or HRP-labeled secondary antibody. In-Cell ELISA characterized primary antibody.Determine background corrected signal and then ratio signal to Janus Green if desired.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) Image the microplate with an IR scanner or develop the HRP labeled microplate and read with a spectrophotometer as appropriate. Dilute the secondary antibody stock to the required concentration (as defined in kit protocol or as determined by user) in 1X incubation buffer and add 100 μL diluted secondary antibody to appropriate wells.Dilute the primary antibody stock to the specified In-Cell ELISA concentration in 1X incubation buffer and add 100 μL to appropriate wells. Add 200 µL of 2X blocking solution to each well.Add 200 µL of 1X permeabilization solution to wells for 30 min.Wash wells with PBS (microplate may be stored at 4☌ at this point).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. 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.
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