Tightly packed monolayers will require some trituration to generate solitary-cell suspensions. Stay away from extreme trituration or shear pressure by resting the pipette suggestion in opposition to the plate edge to keep up viability. If cells never dissociate perfectly, incubation time with dissociation reagent may perhaps should be optimized.
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If cells will not dissociate properly with trituration, select a longer ACCUTASE™ incubation, around 12 minutes. If monolayers don't dissociate following 12 minutes of ACCUTASE™ incubation and trituration, the differentiation was possible unsuccessful.
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There are 2 live-lifestyle morphology indicators for good differentiation and readiness for even more possible characterization. They're:
Going back again to your plate, rinse Each individual well with one mL of FACS buffer and transfer the volume towards the 15 mL tube. Notice: Retain cell suspension on ice just after transfer for the 지방이식 tube until ready to run FACS.
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Notes: Use pipette ideas to Carefully scratch The underside of each nicely in an effort to lift the monolayer off, then dissociate the cell clumps into only one-cell suspension by pipetting up and down 5 - 10 instances.
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