2.1. Cell preparation and measurements

Cells from the 4T1 mouse mammary adenocarcinoma cell line (Caliper Life Sciences, Hopkinton, MA) were frozen after three initial passages. Cells were maintained in media consisting of RPMI (Gibco, Invitrogen Inc., Carlsbad, CA), 10% fetal bovine serum, and 1% penicillin/streptomycin. 67NR cells were purchased from the Animal Model & Therapeutic Evaluation Core (AMTEC), Barbara Karmanos Cancer Institute, Wayne State University. The 67NR cell line is a sister cell population to the 4T1 cell line, both derived from a single spontaneous Balb/cfC3H. The 67NR cells were frozen after three initial passages and were maintained in media consisting of Dulbecco’s Modified Eagle Medium (DMEM, Gibco 11965092), 10% fetal bovine serum, 1% 2mM L-glutamine (Cellgro 25-005-Cl), and 0.1% 1mM Mixed Nonessential Amino Acids (Gibco 11140). All cells were maintained in an incubator at 37∘<!-- ∘ --> C, 5% CO2 , and 95% relative humidity. Prior to imaging, 1×<!-- × -->104 cells were seeded on 25mm diameter No. 1.5 thickness coverslips (Thermo Scientific) in a 6-well plate. Each well was filled with 2mL with the respective media depending on the cell type. Cells were then incubated at 37∘<!-- ∘ --> C, 5% CO2 , and 95% relative humidity for 24 hours.

For imaging, the coverslip was sandwiched in an Attofluor cell chamber, immersed in 1.3 mL of the imaging solution, and finally covered with an 18mm ×<!-- × --> 18mm thickness No. 1 top coverslip (Fisherbrand 12-542A) to ensure planar illumination before being placed on the inverted microscope. The Attofluor chamber was secured to the microscope stage with double-sided tape, which was sufficient to hold the sample in place when switching immersion media so that the same cell can be re-imaged. To switch immersion media, the top coverslip was removed, the original media was aspirated and immediately replaced with the new immersion media, and the top coverslip was replaced. The challenge of changing immersion media with a manual pipette without nudging the sample limited the number of cells for which unmatched and RI-matched data were successfully obtained. Additionally, some image pairs were rejected because the difference in cell shape was judged to be much more extensive than typical. Such changes were assumed to be caused by particularly rapid evolution of the cell or by a disturbance from the manual aspiration step of the media switch.