Macrophage enrichment from induced sputum.
Macrophage enrichment from induced sputum.
复制标题
从诱导痰中富集巨噬细胞。
DOI：
10.1136/thx.2006.073544
复制
发表时间：
2007
期刊：
影响因子：
10
通讯作者：
Alexis,NeilE
中科院分区：
文献类型：
--
作者：
Sikkeland,LivIB;Kongerud,Johny;Stangeland,AstridM;Haug,Terje;Alexis,NeilE
Since induced sputum has become a widely used non-invasive method of recovering cells from the surfaces of the bronchial airways, isolating specific cell populations will be necessary in order to learn more about their specific role in innate immunity and inflammation in the airways. Several studies have demonstrated the ability to conduct ex vivo analyses on sputum cells such as phagocytosis and surface marker measurements, but these have not been performed on isolated cell types. 1–3 This study demonstrates the capability to isolate sputum macrophages from human volunteers in order to advance our understanding of macrophage biology in the airways. To this end, techniques that can enrich and isolate cells without significant activation would prove extremely useful. We compared two common methods for isolating and enriching macrophages in sputum:(1) magnetic bead separation; and (2) Percoll gel density gradient centrifugation. Cell purity and markers of cell activation (mRNA tumour necrosis factor a (TNFa)) and interleukin-1b (IL1b)) were measured at various time points in the isolation process.Nine healthy subjects underwent induced sputum. Sputum collection and sputum processing has been described in detail previously. 4 For measuring natural cell activation over time, we incubated the processed sputum cells for 3 h at 37 C and analysed mRNA TNFa and IL1-b at 0 h (baseline) and 3h. In the positive control experiment we incubated the processed sputum cells with 1 ng/ml LPS (E coli, Sigma). For Percoll (Amersham Biosciences) separation, 600 ml of sputum cell suspension (16106 cells/ml) was layered over Percoll solution (42%) and centrifuged at 560 g for 10 min. Sputum macrophages were removed and incubated at 37 C for 1, 2 and 3 h, respectively, and a pre-incubation sample was also collected. The macrophages were further pelleted and stored at 270 C. For Dynabead separation, CELLection Pan Mouse IgG Kit (Dynal, Norway) was used for immunomagnetic separation of airway macrophages coated with mouse monoclonal IgG2b HLA-DR antibody (Diatec, Norway). Bead coating and cell isolation was performed according to the protocol from the manufacturer. The isolated cells were incubated at 37 C for 1, 2 and 3 h, respectively, and a pre-incubation sample was also collected. The samples were further pelleted and stored at 270 C. Total RNA was extracted (Qiagen) from all the cell samples and reverse transcription was performedIII, Invitrogen. We used pre-developed PCR primers and probes for TNFa and the housekeeping gene PGK (Applied Biosystems). Specific primers and probes were designed for
