The production of antibodies in vitro was introduced by Georges Köhler and César Milstein, who first employed cultures of fused cells to isolate antibodies of defined specificity. In this procedure, termed monoclonal antibody production, antibodies are first produced in mice, and activated B-lymphocytes from the host's spleen (the source of antibodies) are fused with cultured plasma tumor myeloma cells (that do not produce antibodies) from mice of the same strain. The fusion product is a hybrid (termed a hybridoma) of the two cells that continues to grow and divide in culture and produces large amounts of the antibody. Unfused cells and non-productive fusions are eliminated by classical cloning techniques to purify the culture of antibody-producing cells. A hybrid cell line produces only a single type of antibody, which can be used as a selection marker in serial dilution cloning by monitoring the culture medium for secreted antibody at each stage. Ultimately a cell line is established from a single cell (monoclonal) in which all of the cells produce an identical antibody.

In the digital image presented above, epithelial cell tight junctions and nuclear pore complex proteins were simultaneously imaged in MDCK cells with a cocktail of mouse anti-NPCP and rabbit anti-ZO-3 primary antibodies, followed by goat anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa Fluor 568 and Alexa Fluor 488, respectively. Nuclei were counterstained with Hoechst 33258. Note the dispersion of NPCP label in the cell undergoing mitosis at the bottom of the image frame. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.