It truly is significant which the submerged growth cultures in inserts get to 100% confluence just before air-lifting. At one hundred% confluence, the cells will protect the surface area throughout the insert forming a whole, uniform monolayer.
Combine gently prior to transferring cells into a conical tube containing 10 mL of pre-warmed wash medium. Notice: Wash the pipette idea and cryovial While using the wash medium not less than 3 times to assist make improvements to Restoration.
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Operate speedily at this action to pool wells into a 15 mL tube. Be aware: For a sizable-scale dissociation, use a multichannel pipette to pool cells into a sterile reagent reservoir. Include the pooled wells into a 15 mL tube.
Cross-part histology accompanied by hematoxylin and eosin (H&E) staining to assess the thickness on the compact or big airway epithelium
Set the “unstained” tube aside. With the “viability dye” sample, centrifuge the tube at three hundred x g
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Dependant upon the degree of mucus accumulation, a next wash could also be required. See how a mucus wash is performed Within this ALI tradition differentiation video clip (skip to 02:24) >
Notes: STEMdiff™ Kidney Organoid Package includes a different plate structure/culture established-up and is not a normal monolayer culture; kidney organoids protrude through the underlying monolayer society.
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*This is the master resources record for the beneath protocols—all reagents described in Each individual protocol are summarized.
Notes: Use pipette tips to Carefully scratch the bottom of every perfectly as a way to elevate the monolayer off, then dissociate the cell clumps into only one-cell suspension by pipetting up and down five - 10 periods.
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