High-level production of amyloid-beta peptides from IMR-32 cells
OwnerNancy C. Stratman, Donald B. Carterinventors
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Abstract
Methods for producing peptides resulting from the proteolytic processing of amyloid precursor protein (APP). The methods involve high-density plating of IMR-32 cells. IMR-32 cells produced by the methods of the present invention are useful for screening for modulators of peptides resulting from proteolytic processing of APP, e.g., Aβ and sAPPα.
Claims25
A method of culturing IMR-32 cells to increase production of peptides resulting from the proteolytic processing of amyloid precursor protein in the cells, the method comprising steps of:
- (a) seeding a culture vessel with EMR-32 neuroblastoma cells at a density of at least about 5.0×104 cells/cm2 of growth area to about 1.0×106 cells/cm2 of growth area;
- (b) growing the IMR-32 neuroblastoma cells to confluency or near confluency in medium containing serum;
- (c) changing the medium to serum-free medium;
- and
- (d) incubating the IMR-32 neuroblastoma cells in the serum-free medium, wherein, during incubation in the serum-free medium, the IMR-32 cells produce peptides resulting from the proteolytic processing of amyloid precursor protein.
A method of screening for a modulator of proteolytic processing of amyloid precursor protein, the method comprising the steps of:
- (a) seeding a culture vessel with IMR-32 neuroblastoma cells at a density of at least about 5.0×104 cells/cm2 of growth area to about 1.0×106 cells/cm2 of growth area;
- (b) growing the IMR-32 neuroblastoma cells in medium containing serum;
- (c) changing the medium to serum-free medium;
- (d) incubating the IMR-32 neuroblastoma cells in the serum-free medium, wherein the serum-free medium comprises a candidate modulator of proteolytic processing of amyloid precursor protein;
- (e) measuring at least one peptide in the serum-free medium produced by proteolytic processing of amyloid precursor protein by the cells;
- and
- (f) comparing the measurement of step (e) to a control measurement.
A method of detecting an inhibitor of amyloid beta peptide production, the method comprising the steps of:
- (a) seeding a culture vessel with IMR-32 neuroblastoma cells at a density of at least about 5.0×104 cells/cm2 of growth area to about 1.0×106 cells/cm2 of growth area;
- (b) growing the IMR-32 neuroblastoma cells in medium containing serum;
- (c) changing the medium to serum-free medium;
- (d) incubating the IMR-32 neuroblastoma cells in the serum-free medium, wherein the serum-free medium comprises a first concentration of a candidate inhibitor of amyloid beta peptide production;
- (e) measuring a first level of amyloid beta peptide in the serum-free medium containing the concentration of the candidate inhibitor;
- (f) repeating steps (a)-(e) one or more times wherein the concentration of the candidate inhibitor is different than that of the first concentration, wherein a level of amyloid beta peptide is measured for each concentration;
- and
- (g) comparing the level of amyloid beta peptide at two or more concentrations of the candidate inhibitor, wherein a lower level amyloid beta peptide at a higher concentration of the candidate inhibitor indicates an inhibitor of amyloid beta peptide production.
A cell line produced by:
- (a) seeding a culture vessel with IMR-32 neuroblastoma cells at a density of at least about 5.0×104 cells/cm2 of growth area to about 1.0×106 cells/cm2 of growth area;
- (b) growing the IMR-32 neuroblastoma cells in medium containing serum until confluent or near confluent;
- (c) changing the medium to serum-free medium;
- (d) incubating the IMR-32 neuroblastoma cells in the serum-free medium, wherein the IMR-32 neuroblastoma cells secrete amyloid beta peptides into the medium at a rate of at least 1300 pg/ml/day.
Dependencies are inferred from the claim text.