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Cells are cultured (and treated if required) and seeded into a coated 96-well microplate. After fixation and permeabilization, primary antibody is added to the well and is incubated, followed by addition of a labeled secondary antibody. Detection can be colorimetric or fluorescent for a single target using our In-Cell ELISA kits or primary antibodies characterized for In-Cell ELISA. Dual targets can be quantified using IR-conjugated secondary antibodies in our In-Cell ELISA kits. 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.
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. Reagent preparation and hints and tips for a successful assay are included in the appendices.
1. Adherent cell seeding
2. Fix cells to microplate
3. Permeabilize cells
4. Incubation with primary antibody
5. Incubation with secondary antibody
6. Measure signal
7. Whole cell staining with Janus Green (optional)
Analyze data using LI-COR® ICW software, or other suitable data analysis software, such as Microsoft Excel or GraphPad Prism.
Cell seeding density, culture medium and other growth conditions are key to a successful and reproducible experiment. Cell-type specific parameters should be defined by the user.
Cell attachment and fixation
Adherent cells can be grown and treated directly in the assay microplate. Cell attachment can be checked with a microscope. When the cells are fully attached the media can be removed and replaced with media containing the treatment reagent. Culture media containing up to 10% fetal serum does not interfere with the cell fixation and cross-linking to the microplate but may interfere with the treatment reagent.
When using a drug (inhibitor or activator), untreated cells and cells treated with only the drug solvent should be included in the experiment.
Cell seeding density
The cell seeding density of the assay microplate is dependent on the cell type. It depends on the cell size (diameter, in case of the adherent cells) and the abundance of the target protein. As a general guideline, a monolayer is recommended for robust signal. The cell seeding can be determined experimentally by microscopic observation of cell density of serially diluted cells. For adherent cells, prepare a serial dilution of the cells in a microplate (of similar well dimensions) and observe the cell density under a microscope. Working on the high end of this range will generate stronger overall signal and allow detection of small signal increases. E.g., HeLa and HepG2 cells should be seeded from 25,000 to 50,000 cells per well, human fibroblasts (HdFN) from 15,000 to 25,000 cells per well.
It is essential to omit primary antibody in at least one well to provide a background signal for the experiment which can be subtracted from all measured data. This should be done for each experimental condition and with each detector antibody.
Our primary antibodies are supplied with a recommended final concentration for In-Cell ELISA; this is found on each product data sheet.
This assay can also be performed in 384-well microplate format by using ¼ of the volume of reagents and cells, as specified in the above protocol.
|Reagent||Reagent preparation instructions||When to prepare|
|1X PBS||Dilute 45 mL 10X PBS in 405 mL distilled or deionized water. Mix well. Store at room temperature.||At start of experiment|
|1X Wash Buffer||Dilute 625 µL 400X Tween-20 in 250 mL of 1X PBS. Mix well. Store at room temperature.||At start of experiment|
|8% paraformaldehyde solution|
Mix 6.25 mL distilled or deionizd water with 1.25 mL 10X PBS and 5.0 mL 10% paraformaledhyde.
Paraformaldehyde is toxic and should be prepared and used in a fume hood. Dispose of paraformaldehyde according to local regulations.
|Prepare immediately before use|
|1X Permeabilization Buffer||Dilute 250 µL Triton X-100 in 24.75 mL 1X PBS. Mix well.||Prepare immediately before use|
|2X Blocking Solution||Dilute 5 mL 10X blocking solution in 20 mL 1X PBS.||Prepare immediately before use|
|1X Incubation Buffer||Dilute 2.5 mL 10X blocking solution in 22.5 mL 1X PBS.||Prepare immediately before use|
Reagents required for you to design your own In-Cell ELISA experiment are available for purchase in our In-Cell ELISA Support Pack (ab111542), which can be used with adherent and suspension cells. In-Cell ELISA Support Pack (without plates) (ab111541) is also available for purchase.
LI-COR® Odyssey®, Aerius®, IRDye® and In-Cell Western™ are registered trademarks or trademarks of LI-COR Biosciences Inc.
Triton is a trademark of the Dow Chemical Company.
Tween 20® is a registered trademark of Croda Americas.