GFP and human MTGR1 were cloned into the pLEX-307 vector (a gift from David Root, Addgene plasmid 41392). pLEX-307-GFP and pLEX-307-MTGR1 were transfected into HEK 293T cells (ATCC CRL-3216) along with psPAX2 and pMD2.g (gifts from Didier Trono, Addgene plasmids 12260 and 12259). After 48 hours, supernatants were collected and viral particles were concentrated by overnight centrifugation at 9,500g at 4°C. Pelleted lentiviral particles were resuspended in mouse Intesticult media (StemCell Technologies) supplemented with 10μM Y-27632 (Tocris) and mixed with duodenal crypt isolations from WT or Mtgr1−/− mice. Crypt/virus mixtures were incubated for 2 hours at 37°C prior to washing and plating in Matrigel plugs overlaid with ENR media supplemented with CHIR 99021 (3μM, Tocris) and Y-27632. After 4 days, CHIR 99021 and Y-27632 were removed. Viability and gene expression were assessed at day 7 post-plating.
Mouse intesticult media
Manufactured by STEMCELL
Mouse Intesticult media is a specialized culture medium designed to support the growth and maintenance of mouse intestinal organoids. It provides the necessary nutrients, growth factors, and signaling molecules required for culturing and expanding mouse intestinal stem cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using mouse intesticult media
1
Lentiviral Transduction of Intestinal Organoids
2
Lentiviral Transduction of Mouse Intestinal Organoids
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GFP and human MTGR1 were cloned into the pLEX-307 vector (a gift from David Root, Addgene plasmid 41392). pLEX-307-GFP and pLEX-307-MTGR1 were transfected into HEK 293T cells (ATCC CRL-3216) along with psPAX2 and pMD2.g (gifts from Didier Trono, Addgene plasmids 12260 and 12259). After 48 h, supernatants were collected and viral particles were concentrated by overnight centrifugation at 9500 × g at 4 °C. Pelleted lentiviral particles were resuspended in mouse Intesticult media (StemCell Technologies) supplemented with 10 µM Y-27632 (Tocris) and mixed with duodenal crypt isolations from WT or Mtgr1 -/-mice. Crypt/virus mixtures were incubated for 2 h at 37 °C prior to washing and plating in Matrigel plugs overlaid with ENR media supplemented with CHIR 99021 (3 µM, Tocris) and Y-27632. After 4 days, CHIR 99021 and Y-27632 were removed. Viability and gene expression were assessed at day 7 post-plating.
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