Agarose Spin Down Assay: A Technique to Embed Organoids for Histological Analysis
DEŞİFRE METNİ
To process the organoids for histology, remove existing media from the well taking care not to aspirate the basement membrane domes. Add an equal volume of cell recovery solution and incubate the plate for 60 minutes at 4 degrees Celsius.
After the incubation, dislodge the basement membrane dome using a pipette and crush it with the pipette tip. Collect the dissociated dome and the cell recovery solution into a 1.5-milliliter tube. Centrifuge the tube at 300 x g and 4 degrees Celsius for 5 minutes, then remove the supernatant and set it aside.
Save all the supernatant until the final step when the presence of organoids is confirmed. Add the desired volume of cold PBS and gently pipet up and down to mechanically dislodge the pellet without disrupting the organoids. Repeat the centrifugation and remove the supernatant.
Fix the pellet in a matched volume of 4% PFA for 60 minutes at room temperature. After fixation, repeat the centrifugation step and PBS wash. Then, slowly add 200 microliters of warm 2% agarose and immediately but gently detach the cell pellet from the tube wall without disrupting it using a 25 gauge needle attached to a 1-milliliter syringe.
For the agarose spin down method, it is critical to detach the cell pellet from the wall of the tube right after adding agarose using a 25 gauge needle.
Once the agarose has completely solidified, detach it from the tube with a 25 gauge needle attached to a 1-milliliter syringe and transfer it to a new 1.5-milliliter tube. Fill the tube with 70% ethanol and proceed with the conventional protocol for tissue dehydration and paraffin embedding.
