2mg of MSRs were loaded with either 2ug of GM-CSF, 100ug of CpG, or 10ug of Rhodamine-PEI (L25K). The individual vaccine components were then resuspended in 1ml of release media, which is composed of RPMI (Sigma-Aldrich) supplemented with 1% penicillin/ streptomycin and 10% heat-inactivated FBS (Sigma-Aldrich), in low-binding Eppendorf tubes. Release study was then performed in 37 °C under gentle shaking. Periodically, supernatant containing the released component was collected by pelleting the particles at 2000g for 10 min. The release media was subsequently replaced. For analysis, GM-CSF content was determined using ELISA (R&D). CpG content was detected using OliGreen (ThermoFisher)13 (link).
Protocol detail
Vaccine Component Release Kinetics
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Corresponding Organization :
Other organizations : Harvard University, Dana-Farber Cancer Institute, Sungkyunkwan University
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Variable analysis
independent variables
- 2ug of GM-CSF
- 100ug of CpG
- 10ug of Rhodamine-PEI (L25K)
- GM-CSF content
- CpG content
- 2mg of MSRs
- 1ml of release media (RPMI supplemented with 1% penicillin/streptomycin and 10% heat-inactivated FBS)
- 37 °C under gentle shaking
- Periodically collecting supernatant by pelleting the particles at 2000g for 10 min and replacing the release media
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