Do you have a protocol or recommended antibodies to complete ICC staining on my thoroughly differentiated ALI cultures?
The next case in point is for getting ready RNP complexes for 1 response. Modify appropriately according to the volume of reactions needed.
If working with Mild Cell Dissociation Reagent (GCDR), the duration of GCDR therapy must be optimized. Incubate organoids with GCDR for 8 -ten minutes at 37°C and make use of a P1000 pipette to Carefully pipette up and down, and visually inspect if organoids crack up.
If cells will not dissociate properly with trituration, choose a longer ACCUTASE™ incubation, as much as twelve minutes. If monolayers never dissociate immediately after 12 minutes of ACCUTASE™ incubation and trituration, the differentiation was very likely unsuccessful.
The STEMdiff™ system delivers a standardized process for differentiating hPSCs into epithelial cells which might be afterwards cultured in 2D or 3D formats according to the investigation desires.
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Likely back again towards the plate, rinse Every single very well with one mL of FACS buffer and transfer the volume towards the 15 mL tube. Observe: Keep cell suspension on ice after transfer for the tube right up until wanting to operate FACS.
If more substantial clumps are still seen in the solution, return the tube to 37°C for an extra two minutes and repeat the process until eventually organoids have totally broken into single cells.
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Take note: For a really confluent tradition, cultures could be a little bit more clumpy throughout dissociation. To overcome this, Carefully scrape the monolayer that has a pipette idea following including the dissociation reagent to facilitate the dissociation for the duration of incubation.
Of course, you’ll locate the stage-by-action protocol for TEER measurement to evaluate the epithelial barrier integrity in 지방흡입 ALI cultures here.
This can be an conclude-stage protocol built to dissociate PSC-derived kidney organoids into only one-cell suspension and fixation for downstream move cytometry Assessment.
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