1. Procedure a. Long-Term Oxygen Generation i. Mix 25% wt/wt of calcium peroxide powder with Polydimethysiloxane (PDMS) ii.

Load into cylindrical silicone molds in plate (10-mm diameter, 1-mm height). iii. Remove air bubbles removed with a vacuum; iv. Make sure plates were sterilized with ethanol for atleast 30 minutes

v. Attach oxygen sensors to plates vi. Add Dulbecco's Phosphate Buffered Saline (dPBS) vii. Place in a 37O C humidified incubator set at 0.05 mM viii. Monitor the oxygen-generating potential in an incubator using oxygen sensors ix. Set result recording for every 15 seconds. b. Mitigation of Hypoxia-Induced Beta Cell Death via PDMS-CaO2 Disk i. Use 3 105 MIN6 cells seeded within Millicell inserts ii. Place in 24-well plates iii. Place one single PDMS-CaO2 disk beneath the insert, with control cells receiving either no material or a PDMS-only disk.

iv. Incubate at 0.20 mM or 0.010 mM oxygen for 24 h v. Test for oxygen release c. Enhanced Long-Term Proliferation of Cells Cocultured with PDMS-CaO2 Disk i. Use 3 105 MIN6 cells seeded within Millicell inserts ii. Place in 24-well plates iii. Place one single PDMS-CaO2 disk beneath the insert, with control cells receiving either no material or a PDMS-only disk. iv. Incubate at 0.20 mM or 0.05 mM oxygen for 24 days v. Change media after 2 to 3 days vi. Test for oxygen release d. Enhanced Beta Cell Viability for Agarose Constructs Containing PDMS-CaO2 Disk i. Evenly suspend MIN6 cells in 2% agarose (Agarose Type VII) at 37 C ii. Place into molds of disk iii. Centrally place a single plain PDMS disk (control) within construct by adding the disk after half of the cell/agarose mixture was added. iv. Culture at 0.20 mM or 0.05 mM oxygen in the incubator.

2. Material (All cell culture media/reagents purchanged from Invitrogen; chemicals from Sigma-Aldrich) a. Agarose (2%) i. Type VII b. Calcium Peroxide powder

c. Cylindrical silicone molds i.

(10-mm diameter, 1-mm height)

d. Dulbecco's Phosphate Buffered Saline e. Incubator f. MIN6 cells i. (p 3040) g. Plates i. 24-well ii. Quantity: 4 h. Polydimethysiloxane i. 4:1 vol/vol PDMS monomer/platinum catalyst, RTV 615, GE Silicones i. PreSens noninvasive, ruthenium-based oxygen sensors 3. Apparatus a. MicroAmp 24-well Base i. Applied Biosystems MicroAmp Bases are designed to hold MicroAmp
Reaction Tubes in TrayRetainer Sets or MicroAmp Reaction Plates for convenient use in pre- and post-PCR sample processing.

ii. Regulatory Statement 1. For Research Use Only. Not for any animal or human therapeutic or
diagnostic use.

iii. Accessory Type 1. Base iv. Plate Compatibility

1. 24-Well Plates b. Incubator i. Humid CO2incubator 1. Regulatory Statement a. Allows superior control of culture conditions 2. Accessory Type a. Incubator c. Sensor i. Regulatory Statement 1. Oxygen probes measure the partial pressure of both dissolved oxygen
and gaseous oxygen. The oxygen sensor coating is integrated into high grade stainless steel fittings

ii. Accessory Type 1. Sensor

4. Data

5. Sample Calculations a. None

6. Error and Outliers Analysis a. The data points were collected by the oxygen sensors. They were attached to the plates and the data was collected every 15 seconds for a period of 24 hours. b. One set of data was completely removed, due to a human error in the directions. Not enough time was given for cooling and heating of certain solutions.

7. Copy of lab notebook sheets