Ficoll hypaque gradient lymphoprep

Archival paraffin samples of human lung biopsies with diagnosis of TB were collected under a protocol approved by the Ethics Committee of the National Institute for Respiratory Diseases (INER), Mexico. Subjects were of similar socioeconomic status and unrelated to the third generation as determined by a questionnaire. TB patients had evident symptoms (weight loss >10 kg, cough, fever, night sweats for >1 month, or cervical or axillary lymphadenopathy) and chest radiographic findings consistent with recent pulmonary TB. TB was confirmed with a positive sputum acid-fast smear and culture. Fresh blood samples from ATB patients were obtained from patients recruited to the Tuberculosis Outpatient Clinic, INER, Mexico prior to anti-Mtb treatment and did not present co-morbidities such as diabetes, HIV, cancer and Chronic Obstructive Pulmonary Disease. Human peripheral blood mononuclear cells (PBMCs) from ATB patients were isolated by Ficoll Hypaque gradient (Lymphoprep; Axis-Shield POC AS, Oslo, Norway). PBMCs (2.5 × 10⁶ cells/ml) were plated in 24-well plates and incubated at 37ºC in presence of cell wall extract (20 μg/ml) from Mtb H37Rv or Mtb HN878. After a 4-day incubation period, cells were harvested and culture supernatants were used to determinate the levels of IL-22 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).