|Public on Sep 22, 2023
|Subcutaneous myocardial matrix hydrogel injection and nearby dorsal skin
strain: w-sh mice: B6.Cg-KitW-sh/HNihrJaeBsmJ
age: 12-14 weeks old
|Mice received 2x subcutaneous injections in the dorsal region of porcine-derived decellularized myocardial matrix hydrogels.
|Male mast cell deficient mice (w-sh mice: B6.Cg-KitW-sh/HNihrJaeBsmJ, Jackson Laboratory, Sacramento, CA), mast cell engrafted mice and wild-type C57BL6/J were housed till 11-13 week old before performing procedures. Hydrogels were isolated at 11 days post-injection based on latest timepoint for discernible presence hydrogel degradation profile and analyzed for gene expression analysis by Nanostring Mouse Immunology Panel. Mast cell-engrafted mice were created by delivering eight to ten 100 µL subcutaneous injections of bone marrow differentiated mast cells in DMEM totaling 4 million cells. Injections were at evenly spaced intervals throughout the dorsal region of 4-6 week old deficient mice.
|11 days post-injection, mice were euthanized by sodium
|Codeset barcode consisting of 4 fluorophores for identifying each specific hybridized gene probe.
|100 ng of total RNA per sample were labeled with Immunology Panel Reporter CodeSet solution along with relevant buffers and master mix based on manufacturer instructions.
|Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours. Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.
|Immobilized digitial barcode reads from probes hybridized with RNA sample were read on cartridge surface with the Nanostring nCounter Digital Analyzer.
|Samples were processed according to manufacturer instructions. In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit. 70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total. 2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours. Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface. Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
|Data was exported through the manufacturer nSolverTM Analysis Software 4.0 and analyzed through custom R scripts under R versions 4.04 with normalized counts determined by the NanostringDiff package and thresholding outside of one standard deivation from the mean signal from the negative control probes. Specific scripts uploaded to Wang, Raymond M. (2022). NanoString Differential Expression Analysis with NanoStringDiff package (Version 6). Zenodo. 10.5281/zenodo.7190262
|Nov 19, 2022
|Last update date
|Sep 22, 2023
|Raymond Ma Wang
|University of California San Diego
|Sanford Consortium for Regenerative Medicine
|2880 Torrey Pines Scenic Dr
|Nanostring gene expression data, comparison of response to subcutaneously implanted porcine-derived decellularized myocardial matrix hydrogels in mast cell deficient mice compared to deficient mice with mast cell engraftment and wild-type C57BL6/J mice