Appendix A Preparation of Solutions of Fluorescent ROS Sensors
A given dye was warmed up from either a -20 °C freezer or a -4 °C refrigerator to room temperature.
Three of the dyes purchased from Invitrogen were in solution: DHR-123 and HE in dimethyl sulfoxide
(DMSO), APF in dimethylformamide (DMF). Amplex Red powder (Invitrogen) was dissolved in DMSO.
DHF (AnaSpec) was dissolved in methanol. Freshly made phosphate buffered saline (PBS), pH 7.4, was
used to dilute a dye to the working concentration of 100 µM in a 1.5 mL Eppendorf tube. The Eppendorf
tube was covered with aluminum foil to prevent light exposure. If the dye was to be used in subsequent
experiments, it was stored in a -20 °C freezer.
To prepare H2DCFDA for experiments in solution the alkaline hydrolysis protocol as described by
Cohn(Cohn et al., 2008) was used to cleave the diacetate group: (a) 0.0049 g of H2DCFDA was dissolved
in 10 mL methanol, (b) 500 µL of this 1 mM H2DCFDA solution was added to 2 mL of 0.01 N NaOH
and covered with aluminum foil, (c) after 30 minutes at room temperature, 2.5 mL PBS, pH 7.4, was
added to the H2DCFDA/methanol/NaOH solution to make a 100 µM solution.
For cell experiments, H2DCFDA was dissolved in DMSO, and diluted to 100 µM with DPBS.
Appendix B MEF Cell Culture and Handling Protocols
Mouse embryonic cells were used in this study. The tissue culture hood and all reagents and supplies were
wiped down with ethanol. The cells were grown in D-MEM (Quality Biological Inc.) medium with 10%
fetal bovine serum (Cellgrow), 1X Pen-Strep (Gibco), 1X GlutaMAX (Gibco), in 75 cm2 cell culture
flasks (Corning Flask 430641). The flask was removed from the incubator and the cells checked with a
light microscope. In the hood, the cell medium was aspirated, and the cells were washed with 2 mL PBS.
Two milliliters of 0.25% trypsin-EDTA (Gibco) was added to the flask that was then placed in incubator
for 2 minutes. The cells were viewed to ensure they were rounded and releasing from the bottom of flask.
The flask was tapped firmly in the hood and the cells checked with the light microscope again. Two
milliliters of D-MEM medium was added to the flask. The cell solution was carefully mixed. Fifteen
1
milliliters of D-MEM medium was placed in a new flask. The amount of cell solution added to the new
flask was dependent on when the cells were to be split next. If unknown, a small amount of cells was
added (dropwise), checked with scope, and if need be, more cells were added. The new flask was placed
in the 37°C 5% CO2 incubator.
A sleeve of glass bottom culture dishes was opened in the tissue culture hood. Each dish was removed
individually from the sleeve, ensuring the lid was kept in place on the dish. One and a half milliliters of
medium was added to each dish. A 1:8 cell dilution was made in a 15 mL sterile centrifuge tube
(Corning). Five hundred microliters of the 1:8 cell dilution was added to each culture dish, which were
then gently swirled. The dishes were placed in the 37°C 5% CO2 incubator overnight.
2