To determine whether it is possible to isolate sputum cell subpopulations for further molecular biology applications.
Seven (7) patients were studied, three (3) with COPD and four (4) withy Asthma. Sputum was induced via inhalation of a hypertonic saline aerosol, generated by an ultrasonic nebulizer (Ultraneb 2000; DeVilbiss) according to standard methods. The viscid portions of the expectorated sample were separated from the sputum. The cell pellet was processed with magnetic beads columns in order to separate antibody-specific cell subpopulations, (CD45+) for white blood cells (macrophages, eosinophils, neutrophils, lymphocytes) and (HEA) for squamous and non-squamous epithelial cells. The selected cells were stained with a fluorochrome conjugated antibody (FITC) and the subpopulation’s purity was confirmed using flow cytometry.
All seven (7) sputum samples were succesfully processed and separated. The purity of the positive selected samples calculated by flow cytometry was median (min-max) 92% (84-99)%. Furthermore, efficient DNA extraction was performed from each sputum cell subpopulation.
Magnetic labeling of sputum cell subpoppulations with MicroBeads and separation via Macs Columns is an efficient and easy method that could allow to target each cell subpopulation for further molecular analysis.
Separating sputum cell subpopulations via magnetic labeling could enable us to design cell-specific research involved in the pathogenesis of obstuctive airway disease.
K. Samara, None.