METHODS:
J774 cells were cultured in complete medium at 37ºC in a 50 ml culture flask.

Proliferation Assay
J774 cells were treated with Trypsin for 5 min. to remove them from the bottom of the flask. The cells were then scraped using a cell scraper and put into a 15 ml centrifuge tube. They were then spun in the centrifuge for 10 min at 300 x g. The cells were then washed two times by decanting complete medium after spinning, adding new complete medium and spinning again. The cells were then counted on a hemocytometer and resuspended at a concentration of 5 x 105 cells/ml. 100µl of media and 100µl of cells suspension were then added to each well of a 96 well plate. Cells were then treated with media alone, Interferon Gamma (IFN?) and Lipopolysaccharide (LPS), Echinacea "N", Echinacea "J" and Echinacea "H". Cells were left with treatments to incubate for 24 hours before the addition of 5µCi of 3H-thymidine. The cells were left for 4 hours and then harvested onto a cellulose filter. The filters were then individually put into counter tubes and Liquid Scintillation Beta was added to each tube before filters were taken to the counter to determine the amount of disintegration.

J774 Stimulation for ELISA
50,000 cells were added to 36 wells of a 96 well plate. Treatment of IFN, LPS, IFN & LPS and Echinacea (500µg/ml, 50µg/ml, 5µg/ml, 0.5µg/ml, 0.05µg/ml) was added in triplicates and left to incubate for 48 hours. Supernatant triplicates were pooled into 1.5 ml tubes.

ELISA (Enzyme-linked Immunosorbent Assay)
Two 96 well immuno plates were coated with 100 µl of capture antibody and then incubated at 4ºC overnight. The plates were then washed three times with 200 µl PBS/Tween Solution. 200 µl assay diluent was added to each well and incubated at room temperature for one hour. The plates were then washed three times and the supernatants were diluted 1:10 in assay diluent. The top standard was made in assay diluent to 1000 pg/ml and doubling dilutions were also made. 100 µl of each sample and 100 µl of each standard were added to each well that had been coated with the capture antibody. The plates were then incubated at room temperature for 2 hours. They were then washed five times with 200 µl PBS/Tween Solution. 100 µl of the working detector was then added to each well and incubated for an hour at room temperature. The plates were washed seven times and 100 µl of substrate solution was added to each well. The plates were put in a light tight ELISA box for 30 min and then the color production reaction was stopped by adding 50 µl of Phosphoric Acid into each well. The intensity of the color was then measured using a Microplate Reader.

MHC class I, MHC class II, Mac-1 Expression Assay
J774 tumor cells were treated with Echinacea and LPS+IFN and incubated for 48 hours. They were left at 4° for 30 min. before being scraped of the bottom of the culture flask using a cell scraper. Cells and media were transferred to a 15 ml centrifuge tube and spun at 300 x g for 10 min. in the centrifuge. The cells were washed two times by decanting complete medium, adding new complete medium and spinning the cells in the centrifuge. The cells were then counted on a hemocytometer. 100,000 cells from each variant group were put into three 1.5 ml tubes with complete medium. An antibody specific for each molecule was then added to the cells and media. The tubes were left at 4° for another 30 min. before being washed three times. Media was added to the tubes after the last wash and the cells and media were transferred to FACS test tubes. The cells were taken to the FACS machine and analyzed.

Apoptosis Assay
J774 tumors were treated with 5 ml of Trypsin for 5 min. to release the cells from the bottom of the flask. After 5 min. the Trypsin was inactivated with the addition of 5 ml of complete medium. A cell scraper was then used to scrape remainder of cells off the flask. The cells were spun at 300 x g in a centrifuge for 10 min. Cells were then washed twice by decanting complete medium, adding new complete medium, and spinning cells for 10 min. each time. The cells were then counted on a hemocytometer and resuspended in complete medium at a concentration of 5 x 105 cells/ml. 100µl of complete medium and 100 µl of cell suspension were added to each well of a 96 well plate. 2 hours were then allowed for adhesion. Each well was then treated with 5µCi of 3H-thymidine and left for 4 hours. After incubation time, wells were washed by adding 200µl of complete medium to each well three times. Waste was removed into radioactive container, which was disposed of in accordance with radiation protocols. Wells were then treated with media alone, Interferon Gamma (IFNy) and Lipopolysaccharide (LPS), Echinacea "N", Echinacea "J" and Echinacea "H". Wells were harvested after 24 hours onto a cellulose filter. Liquid Scintillation Beta was then added to each filter and then all filters were taken to a counter to determine the amount of apoptosis.