Tightly packed monolayers will require some trituration to make single-cell suspensions. Avoid too much trituration or shear drive by resting the pipette suggestion from the plate edge to keep up viability. If cells will not dissociate well, incubation time with dissociation reagent may perhaps should be optimized.

Most effective outcomes are attained when limiting prolonged cell exposure to ambient temperature situations. Look at keeping unused cells in a very humidified incubator with five% CO2 at 37°C when performing larger experiments.

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If cells tend not to dissociate effectively with trituration, go for a longer ACCUTASE™ incubation, up to twelve minutes. If monolayers tend not to dissociate soon after 12 minutes of ACCUTASE™ incubation and trituration, the differentiation was most likely unsuccessful.

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Heading again on the plate, rinse Each individual very well with 1 mL of FACS buffer and transfer the amount to your fifteen mL tube. Take note: Keep cell suspension on ice following transfer for the tube right up until prepared to run FACS.

Depending upon the amount of mucus accumulation, a second wash could also be needed. See how a mucus wash is performed On this ALI tradition differentiation video clip (skip to 02:24) >

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Notes: Use pipette tips to gently scratch the bottom of each effectively in an effort to carry the monolayer off, then dissociate the cell clumps into only one-cell suspension by pipetting up and down five - ten situations.

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Quickly thaw cells in a very 37°C water tub by gently shaking the cryovial. Clear away the vial when a small frozen cell pellet stays. Note: It is crucial to work speedily in the following methods to ensure substantial cell viability and Restoration.