Independent (Manipulated) Variables

• Cells being tested (hFOB, U2OS, or MIX)
• Status of KM110r Infection
• Incubation temperature
• Time Period Post-KM110r infection

Dependent (Respondent) Variables

• Total cell count in each image
• Number of dead cells in each image
• Presence or absence of “red” signal in RFP channel
• Morphological changes in hFOB cells

Controlled Variables

• All temperature trials were repeated, resulting in over 1000 images and corresponding values.
• hFOB and U2OS cell lines are both bone cells
• Extremely large sample sizes were used (1000 images)
• U2OS and hFOB cell lines were kept under the same conditions in the same incubators
• U2OS and hFOB cell lines were never exposed to any air other than that inside the Cell and Virus Hoods.
• The same inoculum was used for all infections.
• The same formula was used to calculate the ratio at which to split cells for the entire experimentation period
• A Level II Biohazard Hood was used for all splitting and infecting procedures to ensure that no contamination of the cell lines would occur
• When one of each wells (hFOB, U2OS, and MIX) were being infected, their counterparts were mock-infected (all of the same steps were performed except for infection itself).
• The same splitting and infecting procedures were carried out at every stage of the experiment.
• Three fields of view were imaged from each well.
• Each field of view was imaged in bright field, and RFP and GFP where necessary.
• All images were taken at 10x magnification.
• Each channel (bright field, GFP, and RFP) had a unique exposure level, maintained for the duration of the experiment.
• All images were taken with the same microscope.
• All images were contrast-enhanced.
• The images were counted by the same observer.
• The images were viewed on the same computer to be counted.
• A manual cell counter was used; only one person did the manual counting
• The same media (DMEM+10% FBS) was used over the course of experimentation.
• The same concentration of Trypsin (1x) was used over the course of experimentation.