To begin this procedure, cannulate the trachea by inserting the silicone tube, and fixing it with a clamp parallel to the tube so that the clamp squeezes the tissue together alongside the silicone tube without pinching it off. Close all other bronchi, blood vessels, and injuries with clamps so that no agarose can leak out during the filling procedure.
Good anatomical skills are needed to cannulate the main bronchus. The lungs should not be expanded too much otherwise the tissue will distract, and if the tissue is not filled enough, it will be impossible to make any PCLS because the tissue is too soft.
Then, mix an equivalent volume of 3% low-gelling agarose with culture medium in a beaker, and instill the mixture into the lung using a 50-milliliter syringe. Prior to refilling the syringe with medium, clamp shut the catheter with fingers or a clamp to avoid air bubbles and agarose-free flux. Cut the lung tissue into 3 to 5-centimeter slabs.
Fill the tissue slicer with 400 milliliters of ice-cold EBSS. Immediately, cut the cylindrical tissue cores out of the lung slabs using a semi-automated screwdriver with a coring tool. Afterward, transfer the tissue cores into the tissue holder of the tissue slicer. Place the weight on top of the tissue core and start slicing the tissue core into PCLS. Medium should be drained out of the tissue slicer into a beaker by opening the clamp of the glass cylinder.
Next, transfer the slices from a beaker to the Petri dish with culture medium. Following that, place the Petri dish into an incubator and allow the medium to warm up prior to the washing steps. Then, transfer the lung slices carefully into a 24-well culture plate with a minimum of 500 microliters of culture medium for two slices per well.
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