U2OS cells are moderately differentiated bone cancer (osteosarcoma) cells genetically recombined with green fluorescent protein (GFP), which is found in a jellyfish that lives in the cold waters of the north Pacific ocean. The jellyfish contains a bioluminescent protein—aequorin—that emits blue light. The green fluorescent protein converts this light to green light, which is what we actually see when the jellyfish lights up. Solutions of purified GFP look yellow under typical room lights, but when taken outdoors in sunlight, they glow with a bright green color. The protein absorbs ultraviolet light from the sunlight, and then emits it as lower-energy green light. This property makes GFP an excellent marker under immunofluorescence.

hFOB cells are normal undifferentiated pre-cursor bone cells containing a temperature sensitive mutation (tsA58)  that drives differentiation in response to temperature change.

Oncolytic virus KM110r is a genetically engineered double-mutant virus that combines the n212 ICP0 mutation with the V422 VP16 mutation. It is coalesced with a bioluminescent protein called red fluorescent protein (RFP).

The cells were cultured in 6-well dishes which contained two wells each of hFOB, U2OS, and MIX co-culture (50% U2OS, 50% hFOB) seeded at concentrations of 1.3x10⁵ cells/mL. One of each group was infected using KM110r at a multiplicity of infection (MOI) of 1, while their counterparts were mock-infected as the negative-control. Three experimental trials were conducted at incubation temperatures of 34°C, 37°C, and 39°C to induce genetic differentiation in the hFOB cells. Each temperature trial was fully repeated to ensure reliability among the data.

Each experimental trial employed immunofluorescent microscopy to conduct a proliferation assay over the course of four time periods spanning a five day infection since full differentiation is expected in this time period. During each time period three areas in each of the wells were imaged in bright field light, GFP and RFP channels. Approximately forty images were taken each day, totalling almost one-thousand images over the course of experimentation. The cells in each image were subsequently counted and morphologically analyzed.

During the 6 month experimentation period, each cell line (hFOB and U2OS) required splitting for maintenance. Maintenance splitting needed to be carried out every 2-3 days.