Tightly packed monolayers would require some trituration to build solitary-cell suspensions. Keep away from extreme trituration or shear force by resting the pipette idea versus the plate edge to take care of viability. If cells will not dissociate very well, incubation time with dissociation reagent may well must be optimized.
When the operate is finish, retrieve the Supply Cartridge from the instrument. The cell sample need to be at the bottom or side of the collection tube. Be aware: It is suggested to spin down the Shipping Cartridge in the mini-centrifuge for your several seconds for comprehensive quantity Restoration.
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Established the “unstained” tube aside. For your “viability dye” sample, centrifuge the tube at 300 x g
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Notes: STEMdiff™ Kidney Organoid Package involves a unique plate format/lifestyle set-up and isn't a normal monolayer culture; kidney organoids protrude with the underlying monolayer lifestyle.
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Note: For an extremely confluent lifestyle, cultures could be a little bit more clumpy throughout dissociation. To overcome this, gently scrape the monolayer that has a pipette stem cell clinic idea after adding the dissociation reagent to facilitate the dissociation in the course of incubation.
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Clear away supernatant and resuspend cells in FACS buffer. Notice: It's important to quench the dissociation reagent by utilizing the very same or double the volume of your dissociation reagent.