As previously described major normal verbal keratinocytes were gathered from normal gingival tissues and cultured. Muscle selection was approved by the UCSF Committee on Human Research and permission was obtained from patients. NOK were cultured in Defined Keratinocyte Serum free media. All three cell lines were formulated with100 g/mL streptomycin sulfate, 100 U/mL penicillin and 25 g/mLfungizone and developed at 37 C with 5% CO2. We examined the existence of cannabinoid natural product library receptors on human oral cancer cells using immunofluorescence. HSC3 cells were grown on cover slips over night, then washed with PBS and fixed in cold acetone for 10 minutes. Incubation with rabbit polyclonal anti CBr2 and primary goat polyclonal anti CBr1 antibody, was done at 4 C over night. The cells were incubated with the secondary anti goat IgG FITC and anti rabbit Texas Red conjugated antibody for 1-hour at room temperature. The nuclei were stained with Hoechst 33342. Cover slips were mounted on in Gel Mount and visualized on a Nikon Eclipse E600 microscope using epi uorescence. The images were taken and analyzed with a RT Spot Camera and RT Spot Software. Controls involved the omission of the main antibodies for CBr2 and CBr1 during incubation. We used american blot Inguinal canal to ensure CBr1 and CBr2 appearance. NOKs, SCC9 and hsc3 were lysed in Nonidet P 40 lysis buffer. Protein concentration was based on BCA Protein Assay Kit. Proteins were separated by SDS polyacrylamide gel electrophoresis and utilized in a nitrocellulose membrane using a semi dry blotting apparatus. The membranes were developed using ECL Chemiluminescence Kit and bands were detected by contact with X-ray film. The blots were quantified and assigned rvu using an image analysis program. We examined the consequences of cannabinoid receptor agonists on human oral cancer cell proliferation using the MTS assay. HSC3 cells were plated over a 96 well Ivacaftor ic50 plate. The cells were serum starved for 24 hours to allow synchronization. Serial dilutions of WIN55,212 2, ACEA, and AM1241 were prepared in 0. 2000 DMSO/water and brought to each group. Vehicle served as the control. The plates were incubated and assayed every 24 hours for a period of time of 4 days. During the time of analysis, 20 l of MTS reagent was put into each well. Plates were incubated for 2 hours at night. Absorbance was recorded utilizing a microplate reader calibrated to 490 nm. The oral cancer mouse model was produced by inoculating HSC3 cancer cells to the hindpaw of mice as previously described. Tests were conducted on female Foxn1nu, athymic, immuno-compromised rats ranging from 4 C5 days old and weighing 20 C25g during the time of inoculation. Mice were housed in a temperature controlled room over a 12:12 h light cycle with advertisement libitum access to water and food.