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Cell Biology JoVE (Open Access) Citable · DOI

Methodology for Sputum Induction and Laboratory Processing

DOI: 10.3791/56612-v
What you'll learn
  • ✓ Perform sputum induction using standardized nebulization technique
  • ✓ Process induced sputum samples for cellular analysis
  • ✓ Prepare differential cell counts and cytospin slides
  • ✓ Collect sputum fractions for downstream molecular analysis
Protocol

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

Difficulty
intermediate
Total time
~1–2 hours per subject (induction ~20–30 min; processing ~30–60 min)
Biosafety
BSL-2

Steps

1
Induce sputum using nebulized saline

Administer nebulized hypertonic saline to the subject to stimulate sputum production. Collect expectorated sputum in sterile containers following standardized timing protocols.

▶ 01:10
2
Process sputum for cellular separation

Treat collected sputum with dithiothreitol (DTT) to reduce mucus viscosity, filter through gauze, and centrifuge to separate cells and supernatant fractions.

▶ 05:27
3
Perform cell counts and prepare cytospins

Count viable cells using a hemocytometer and prepare cytospin slides with staining (e.g., Diff-Quik) for microscopic differential cell analysis.

▶ 10:34
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