pool 2 of E17.5 WT embryos
Mus musculus
Black Swiss background Protocols: Remove embryos and place in cold PBS. Dissect out pancreas from each embryo and place in cold 1X DPBS -/-. Use fluorescent dissecting scope to determine which pancreases are EGFP+ and place them in RPMI + 2% FBS until genotypes are determined. After determining genotypes, pool together WT/EGFP+ and Mut/EGFP+ embryos in 2mL eppendorf tubes and add 1 mL of cold 1X DPBS -/-. Use dissecting scissors or tweezers to chop up Pancreatta to about 1 mm pieces. Spin at 2500 rpm for 5-10' and remove supernatant. Resuspend in 1 mL Accumax and add 5 ul (5 mg/ul) DNaseI. Incubate at 37 degrees for 30-35'. Time will vary with number of Pancreatta and age of embryos. Agitate tube every 5-10 minutes by inverting the tube or pipetting up and down. Be sure to use a fresh pipette tip for each genotype and do not let the tissue get stuck in the tip (as the pancreas can be very sticky). After dissociation, add 1 mL RPMI 1640 with 2% FBS and an additional 5 ul of DNaseI. Spin 2500 rpm for 5' at 4 degrees (keep cold). Remove supernatant and add 1 mL 1X DPBS -/- to wash and remove phenol red from RPMI. Spin at 2500 rpm for 5'. Remove supernatant and resuspend in 250 ul/pancreas (up to 1 mL) 1X DPBS -/-. Filter through blue cap falcon filter tube. Add 1 ul DAPI just before going to the sorter. Vortex sample to mix in DAPI. Keep samples on ice. For sorting we used mCherry chanel, meaning that the cells sorted will be Ghrelin-expressing and also Ghrelin descendants. Cell purity > 99%.
ENA-FIRST-PUBLIC
2015-02-25T17:03:21Z
ENA-LAST-UPDATE
2018-03-08T23:42:40Z
External Id
SAMEA3270052
INSDC center name
Columbia Unversity
INSDC first public
2015-02-25T17:03:21Z
INSDC last update
2018-03-08T23:42:40Z
INSDC status
public
Submitter Id
E-MTAB-3353:pool 2 of E17.5 WT embryos
age
E17.5
broker name
ArrayExpress
common name
house mouse
sample name
E-MTAB-3353:pool 2 of E17.5 WT embryos
scientific_name
Mus musculus
strain
Ghrltm1(Cre-eGFP)Suss; B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J