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Best final results are attained when limiting prolonged cell publicity to ambient temperature circumstances. Take into consideration retaining unused cells in a very humidified incubator with 5% CO2 at 37°C when executing much larger experiments.
If using Light Cell Dissociation Reagent (GCDR), the period of GCDR treatment will have to be optimized. Incubate organoids with GCDR for eight -ten minutes at 37°C and utilize a P1000 pipette to Carefully pipette up and down, and visually inspect if organoids split up.
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Diligently clear away the supernatant with out disturbing the organoid pellet. Increase 0.5 mL of ideal one-cell dissociation reagent towards the tube. Distinct incubation tips for various dissociation reagents are mentioned under:
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Carry out cell counts making use of an automatic cell counter. Take note: Reduced viability can be optimized through the duration of collagenase or ACCUTASE™ incubation. It will also be optimized by minimizing the number of triturations.
This is an finish-stage protocol created to dissociate PSC-derived kidney organoids stem cell clinic into an individual-cell suspension and fixation for downstream circulation cytometry Examination.
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