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The in vitro differentiation of human pluripotent stem cells (hPSCs) into distinctive cell and tissue styles permits the examine of human biology without the require for Major tissues or in vivo versions.
If cells don't dissociate nicely with trituration, opt for a longer ACCUTASE™ incubation, up to twelve minutes. If monolayers never dissociate after twelve minutes of ACCUTASE™ incubation and trituration, the differentiation was probable unsuccessful.
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Dependant upon the degree of mucus accumulation, a 2nd clean could also be required. See how a mucus wash is performed Within this ALI tradition differentiation video clip (skip to 02:24) >
Certainly, you’ll find the actions to perform an ICC staining on the epithelial cells cultured for the ALI With this protocol. Here is a summary of antibodies that may be used for the characterization of airway cultures:
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When building tutorial RNAs, it is suggested to select sequences that align While using the transcription begin web-site of your gene of interest in which feasible.
Use cell counts to individual Stem cell the sample into two tubes – “unstained” and “with viability dye”.
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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.