Immunoprecipitation (IP) of HL60 Cell Lysate

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. It is also used to analyze protein fractions separated by other techniques such as gel filtration or density gradient sedimentation. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or intrinsically labeled cells, or in vitro-translated proteins. This unit describes a wide range of immunoprecipitation techniques, using either suspension or adherent cells lysed by various means.Usually, plasmas transfected cells or natural cells are ideal sample for immunoprecipitation.

HL-60 cell line was derived from a 36-year-old woman with acute promyelocytic leukemia at the National Cancer Institute. HL-60 cells are predominantly a neutrophilic promyelocyte (precursor) which has been used for laboratory research on how certain kinds of blood cells are formed. The doubling time of HL-60 is about 36–48 hours which proliferates continuously in suspension culture in nutrient and antibiotic chemicals. 

Proliferation of HL-60 cells occurs through the transferrin and insulin receptors, are expressed on cell surface. The requirement for insulin and transferrin is absolute, as HL-60 proliferation immediately ceases if either of these compounds is removed from the serum-free culture media. With this line, spontaneous differentiation to mature granulocytes can be induced by compounds such as dimethyl sulfoxide (DMSO), or retinoic acid and other compounds like 1,25-dihydroxyvitamin D3, 12-O-tetradecanoylphorbol-13-acetate (TPA) and GM-CSF can induce HL-60 to differentiate to monocytic, macrophage-like and eosinophil phenotypes, respectively.

The HL-60 cultured cell line provides a continuous source of human cells for studying the molecular events of myeloid differentiation and the effects of physiologic, pharmacologic, and virologic elements on this process. HL-60 cell model was used to study the effect of DNA topoisomerase (topo) IIα and IIβ on differentiation and apoptosis of cells and is peculiarly useful in dielectrophoresis studies, which require an aqueous environment with suspended and round cells.