We highlight the protocol using Polymorphprep (PMP) due to its affordability. In addition, there are multiple methods to isolate neutrophils from the blood of human donors.
#SA COLOR FINESSE 3 SERIAL FULL#
1) Growing epithelial cells on filters for two weeks at air-liquid interface (ALI) for full differentiation 2) Isolating primary neutrophils from human blood 3) Flipping the epithelialized filters to enable transmigration 4) Transmigration of the neutrophils and subsequent conditioning into airway-like neutrophils and 5) Use of transmigrated neutrophils in a bacteria-killing assay.Īlternatives: We have also used this model with sputum from patients with asthma, chronic obstructive pulmonary disease (COPD) and tracheal aspirates from pediatric patients with acute respiratory distress syndrome (ARDS) ( Grunwell et al., 2019). The procedure is divided into five main steps (Graphical Abstract). The objective of the below protocol is to condition primary blood neutrophils to an airway-like phenotype via transmigration in an organotypic model of human airways with apically applied sputum supernatant from patients with cystic fibrosis (CFASN). Confirm that you are allowed, and able, to process primary patient material with your local Medical Ethics Committee and Internal Review Board (IRB) and obtain informed consent from the donors for the use of 1) blood and 2) sputum. This protocol details the use of primary patient material.
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