Modulation of inflammatory cytokine production by Perna was determined by cytokine bioassays [
25‐
28]. Supernatants of Perna-treated and untreated cytokine producing cell lines (producers) were added to indicator cell lines specifically sensitive to one of the cytokines. Proliferation or inhibition of these indicator cell lines signals the extent of cytokine secretion by the producer cell lines. The optimum cell number per well for each cell line was determined by titration. The producer cell lines which grow in cell suspension, THP-1, Jurkat E6-1, EL-4 and U-937, were plated on 48 well plates at a concentration of 5e
6 cells/well, whereas the adherent producer cell line, LS174T, was plated at a concentration of 5e
5 cells/well. Suspension responder cell lines (CTLL-2 and 7TD-1) were added at a concentration of 1e
6 cells/well in a 96 well plate, whereas the adherent responder cell lines, L-929 and A375.S2, were plated at concentrations of 3e
4 cells/well and 4e
4 cells/well respectively. THP-1 cells, which secrete tumor necrosis factor-alpha (TNF-α) when stimulated with lipopolysaccharide (LPS), were treated with varying concentrations (containing 0 μg, 5 μg, 10 μg, 15 μg, 20 μg, and 25 μg protein) of Perna for a period of 24 hours at 37°C in a humidified 5% CO
2 incubator. Supernatants from these cells were then added to the indicator cell line L929 that is sensitive to TNF-α and incubated for 24 hours at 37°C in a humidified 5% CO
2 incubator [
29‐
31]. After incubation, MTS-PMS solution, (Cell Titer 96 Aqueous Kit, Promega, Madison, WI) was added to each well as recommended by the manufacturer [
32]. Detection of cell proliferation or inhibition is based on a colorimetric assay system utilizing the tetrazolium reagent, MTS. MTS is reduced to a water soluble formazan dye, via the alternate electron acceptor PMS (phenazine methosulphate) in the mitochondria of living cells. Samples are read at 4 hours using a BIO-RAD benchmark microplate reader at 490 nm. The amount of color produced is directly proportional to the number of viable cells. The experiments were carried out in triplicate and repeated three to six times for each cytokine. Similarly, U-937 cells which secrete Interleukin-1 (IL-1) constitutively were treated with Perna and the supernatants added to the sensitive cell line A375.S2 [
33,
34]. Likewise, Jurkat E6-1 and EL-4 mouse thymoma cells which when stimulated with Ionomycin and phorbol myristate acetate (PMA) respectively secrete IL-2, were treated with Perna and the supernatants were added to the responder cell line CTLL-2 [
35‐
38]. Finally, LS174T colon adenocarcinoma cells that constitutively secrete IL-6 were treated with the Perna extracts and the supernatants added to the responder 7TD1 cell line [
39‐
41].