IntestiCult™ Organoid Growth Medium (Mouse)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Replace media every 4 days Collect organoids after 14 days

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Replace media every 4 days Collect organoids after 14 days

Publication protocol

Gastric organoids: The mouse stomach was cut into 2–3 mm pieces on ice with cold PBS and then incubated in 1 ml of 5 mM EDTA for 30 min at 37°C with shaking and centrifuged at 1,500 rpm for 5 min at 4°C. The cell suspension was filtered through a 70 μM filter. This was followed by centrifugation of the crypts fraction at 1,500 rpm for 10 min at 4°C. The pellet was resuspended in 5 ml ice-cold PBS. The crypts were centrifuged and the pellet was suspended in 50 μl Matrigel (BD Biosciences) and plated in pre‐warmed 24‐well plates. The matrigel was allowed to solidify for 20 min in the tissue culture incubator, 500 μl mouse basal medium (STEMCELL, 06000, Cambridge, MA) was overlaid, and media was replaced every 4 days. Organoids were collected after 14 days, fixed in 4% PFA for 20 min, washed in 1× PBS and re‐suspended in 30 μl of Histo-gel (Richard-Allan) and mixed with the cell pallet. Carefully, we removed the histo-gel dome from the tube, placed the dome into a cassette and placed into 70% ethanol then processed for paraffin embedding. Paraffin-embedded organoids were pretreated at 65°C for 2h, following by deparaffinization procedures. Antigen retrieval was performed using a solution of 10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0. Slides were then incubated for primary antibody overnight at 4°C followed by washes with PBST ( PBS plus 0.1% Tween 20) three times, and then incubated with both Alexa Fluor goat anti-rabbit IgG and Alexa Fluor goat anti-mouse IgG (1:800) (Invitrogen) at room temperature for 2 h. Images were acquired using an inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.


Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing 3D Cell Culture Media Mouse gastric cancer organoids using IntestiCult™ Organoid Growth Medium (Mouse) from STEMCELL technologies.

Manufacturer protocol

Download the product protocol from STEMCELL technologies for IntestiCult™ Organoid Growth Medium (Mouse) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media Mouse gastric cancer organoids using IntestiCult™ Organoid Growth Medium (Mouse) from STEMCELL technologies. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.