1 March 2002

Reagents:

• DPBS: Dulbecco’s Phosphate Buffered Saline, calcium/magnesium free (BioWhittaker #17-515F)

• DTT: Sputolysin (Calbiochem #560000, 20x 10ml). Dilute the contents of 1 vial to 100ml with sterile distilled water. Swirl gently to dissolve any crystals; filter if necessary. Diluted solutions, stored under N₂, are stable for 48 hours at 2-8°C. After opening, vials with undiluted concentrate can be stored under N₂at 2-8°C.

Protocol:

Process sputum within 2 hours of collection.

Keep sputum on ice until ready to process.

Select opaque/dense sputum portions that are different from saliva and free of squamous cell contamination for processing.

Weigh remaining sputum sample.

Add freshly prepared DTT equal to 2x weight of sputum portion.

Incubate 15 minutes at 37°C with frequent shaking.

Vortex every 5 minutes for 15 seconds.

Dilute with equal volume of DPBS.

Filter through 48 µm pore nylon gauze mesh filter.

Take sample for hemocytometer: obtain total cell count and viability.

Spin cells at 2000 rpm for 10 minutes (4°C).

Collect supernatant and store at -80°C.

Resuspend cell pellet in DPBS at 10⁶cells/ml.

Make cytospins with 60,000 to 120,000 cells per slide.

Fix and stain (May-Grunwald Giemsa, Wright, and/or Romanowski stains).

Use remaining cells for culture assays.

References:

1. Pin et al. (1992)Am Rev Respir Dis145:1265-1269.

2. Pin et al. (1992)Thorax47:25-29.

3. Pizzichini et al. (1996)Am J Respir Crit Care Med154:308-317.

4. Pizzichini et al. (1997)Am J Respir Crit Care Med155:1501-1508.

5. Lensmar et al. (1998)Eur Respir J12:595-600.

6. Ward et al. (1999)Clin Exp Allergy29:248-252.

7. Rutgers et al. (2000)Eur Respir J15:109-115.

8. Purovicki et al. (2000)Eur Respir J16:242-246.

9. Alexis et al. (2000)Clin Immunol97:21-32.

10. Zeibecoglou et al. (2000)J Allergy Clin Immunol106:697-704.