Organoid isolation, passage and cryopreservation were performed as previously described13 , 18 (link). Cre recombination in vitro was induced by adding 3ul of 2×1010 PFU/mL Adenoviral-Cre (University of Iowa Gene Transfer Vector Core) to 250ul of Matrigel containing freshly isolated crypts and plated as above. Mouse Tgfb1 (eBioscience, 14-8342-62) was used at 10ng/ml. Nutlin (Selleck Chemicals, S1061) was used at 10 μM. DNA, Protein and RNA isolation, and qRT-PCR analysis of organoids was performed as previously described16 (link), 18 (link). Prior to transplantation, organoids were amplified in 200 μl of Matrigel plated in one well each of a 6-well dish and supplemented with 3 milliliters of colon organoid growth media13 . For conversion of organoids into 2-D tissue monolayer conditions, organoids were passaged as previously described but resuspended into DMEM (+Pen/Strep +10% Fetal Bovine Serum) and plated onto a 10 cm sterile tissue culture dish (Falcon, 353003), and passaged 1:5 every 3–5 days.
Nutlin
Manufactured by Selleck Chemicals
Nutlin is a lab equipment product manufactured by Selleck Chemicals. It is a small molecule that functions as an inhibitor, with its core purpose being to disrupt the interaction between the proteins p53 and MDM2.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using nutlin
1
Organoid Isolation, Passage, and Cryopreservation
2
Organoid Isolation, Passage, and Cryopreservation
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Organoid isolation, passage and cryopreservation were performed as previously described13 , 18 (link). Cre recombination in vitro was induced by adding 3ul of 2×1010 PFU/mL Adenoviral-Cre (University of Iowa Gene Transfer Vector Core) to 250ul of Matrigel containing freshly isolated crypts and plated as above. Mouse Tgfb1 (eBioscience, 14-8342-62) was used at 10ng/ml. Nutlin (Selleck Chemicals, S1061) was used at 10 μM. DNA, Protein and RNA isolation, and qRT-PCR analysis of organoids was performed as previously described16 (link), 18 (link). Prior to transplantation, organoids were amplified in 200 μl of Matrigel plated in one well each of a 6-well dish and supplemented with 3 milliliters of colon organoid growth media13 . For conversion of organoids into 2-D tissue monolayer conditions, organoids were passaged as previously described but resuspended into DMEM (+Pen/Strep +10% Fetal Bovine Serum) and plated onto a 10 cm sterile tissue culture dish (Falcon, 353003), and passaged 1:5 every 3–5 days.
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