Live Cell Imaging of Confluent Cells and Xenograft Tumors

Confluent cells were imaged on 35-mm glass bottom dishes at 37°C and 5% CO₂ using a Leica DMI 6000 SP8 confocal microscope or an Olympus FV1000 confocal microscope with a SIM scanner (Serrels et al., 2009 (link)). For photobleaching (FRAP, FLIP) experiments in live xenograft tumors, mice were anesthetized using a combination of 1:1 hypnorm - H₂O + 1:1 hypnovel - H₂O, and the subcutaneous tumor was exposed surgically via a skin flap procedure (Serrels et al., 2009 (link)) on a 37°C heated stage. Mice were sacrificed within 4 hr of imaging.
Detailed protocols can be found in the Supplemental Experimental Procedures.

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Erami Z., Herrmann D., Warren S.C., Nobis M., McGhee E.J., Lucas M.C., Leung W., Reischmann N., Mrowinska A., Schwarz J.P., Kadir S., Conway J.R., Vennin C., Karim S.A., Campbell A.D., Gallego-Ortega D., Magenau A., Murphy K.J., Ridgway R.A., Law A.M., Walters S.N., Grey S.T., Croucher D.R., Zhang L., Herzog H., Hardeman E.C., Gunning P.W., Ormandy C.J., Evans T.R., Strathdee D., Sansom O.J., Morton J.P., Anderson K.I, & Timpson P. (2015). Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue. Cell Reports, 14(1), 152-167.

Publication 2015

FV1000 confocal microscope

Cells Confocal microscope Dishes Experiments tumors Flap Hypnorm Mice Skin Surgically Tumor Xenograft

Corresponding Organization : St Vincent's Clinic

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Variable analysis

independent variables

Photobleaching (FRAP, FLIP) experiments in live xenograft tumors

dependent variables

Measured outcomes of photobleaching experiments

control variables

Temperature maintained at 37°C
5% CO2 environment
Anesthesia using a combination of 1:1 hypnorm - H2O + 1:1 hypnovel - H2O
Subcutaneous tumor exposed surgically via a skin flap procedure
Mice sacrificed within 4 hr of imaging

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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