Cell type

Primary cells vs. an established cell line
Primary cells are often difficult to culture. Phenotype and viability may change within a few passages. Established cell lines are typically easier to culture and usually maintain viability and phenotype over numerous passages.

Number of cells per well

Cell passage number

Cells can change their responsiveness over passage number. Primary cells in particular have a tendency to change over passage number. Receptor number may be dependent on passage number or condition that the cells are maintained under.

Growth medium

Each cell type has specific medium optimized for growth. This information can be found in ATCC or in publications.

Growth serum type

Serum type is usually specified in the literature or in ATCC. Dextran Charcoal (DCC) treated serum removes proteins, which may interfere with the assay. Cells may require Horse serum in addition to Fetal bovine serum.

Growth serum concentration

Serum is almost always required for growth conditions unless the medium has been optimized for serum free conditions. Ranges are from 1-15% serum concentrations. Serum free conditions are often used to prevent interference with compounds binding to protein or to increase the stimulation of the inducer.

Treatment medium

Medium being used during the treatment can differ from that used in growth medium.

Treatment serum type

Serum type is usually specified in the literature, by ATCC, or from the originator of the cell line. However, compound testing can be done in serum free conditions.

Treatment serum concentration

Serum free conditions are often used during treatment of compounds once cells have been established in serum to remove effects of serum. Low concentrations of BSA are often substituted to prevent non-specific protein binding from occurring.

Incubation time for pretreatment

Dependent on response desired for assay and biological relevance.

Treatment incubation time

Dependent on desired assay response and biological relevance.

Plate type

Plate type is assay dependent. Fluorescent assay:black or white; Luminescent: white or black; Radioactive:Plates containing scintillant coated on the bottom or on the bottom and sides, or filter plates for filter binding. Colorimetric: clear tissue culture treated plates for assays measuring proteins in a cell culture supernatant like an ELISA, the supernatant is transferred to the ELISA plate.

Coating type

Some cells adhere better with some type of coating, reducing variability and enhancing the response. The coating can be poly-D-lysine, fibronectin, collagen or other matrix, that enhances cell attachment to the plate.

pH

Neutral

Inducer/Inhibitor concentration

Dependent on experimental conditions.

Radioactive tracer concentration

Based on optimal signal window obtained during assay optimization.

Addition time of radioactive tracer

Based on optimal signal window obtained during assay optimization and biological relevance. The simplest method is to add the radioactive tracer at the same time as the inducer and then measure the response over a period of time post induction.

Type of radiation

¹⁴C is usually needed for plates containing scintillant coated on the bottom to get an optimal signal window. Other types of radioactive tracers may be used for other assay platforms.