Tightly packed monolayers would require some trituration to create single-cell suspensions. Keep away from excessive trituration or shear power by resting the pipette tip against the plate edge to take care of viability. If cells never dissociate perfectly, incubation time with dissociation reagent may perhaps need to be optimized.
As soon as the run is full, retrieve the Supply Cartridge from the instrument. The cell sample ought to be at the bottom or side of the collection tube. Note: It is recommended to spin down the Delivery Cartridge inside a mini-centrifuge to get a few seconds for complete quantity Restoration.
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It is vital to quench the dissociation reagent employing FACS buffer or prevent medium, by introducing a minimum of the identical or double the volume from the dissociation reagent.
Cross-section histology followed by hematoxylin and eosin (H&E) staining to evaluate the thickness of the tiny or substantial airway epithelium
For greatest results, the full volume of cargo added mustn't exceed 10% of your reaction volume. Minimizing the reaction quantity to under eighty µL may possibly lead to reduce enhancing efficiencies and cell recoveries.
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Here's a listing of posted literature that cites culturing of nasal epithelial cells using PneumaCult™ culture media:
In the event cell clumping is noticed soon after thawing cryopreserved cells, it is suggested to filter aggregated suspensions through a 37 µm cell strainer (e.g. Catalog #27250) for exceptional outcomes. In cases like this, the operate time about the CellPore™ Transfection System could must be increased to 10 seconds.
To evaluate the regional specificity in the little vs large airway, you could complete the next assays:
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Notes: Use pipette tricks to gently scratch the bottom of every very well so that you can lift the monolayer off, then dissociate 자가지방이식 the cell clumps into only one-cell suspension by pipetting up and down 5 - ten periods.
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