NBBAL and BAL samples were filtered through a 70-μm cell strainer, pelleted by centrifugation at 300 rcf for 10 min at 4°C, followed by hypotonic lysis of red blood cells with 2 ml of BD PharmLyse reagent for 2 minutes. Lysis was stopped by adding 13 ml of MACS buffer (Miltenyi Biotech). Cells were pelleted again and resuspended in 100 μl of 1:10 Fc-Block (Human TruStain FcX, Biolegend) in MACS buffer, and a 10-μl aliquot was taken for counting using K2 Cellometer (Nexcelom) with AO/PI reagent. The volume of Fc-Block was adjusted so the concentration of cells was always less than 5×107 cells/ml and the fluorophore-conjugated antibody cocktail was added in 1:1 ratio (Extended Data Table 3). After incubation at 4°C for 30 minutes, cells were washed with 5 ml of MACS buffer, pelleted by centrifugation, and resuspended in 500 μl of MACS buffer with 2 μl of SYTOX Green viability dye (ThermoFisher). Cells were sorted on a FACS Aria III SORP instrument using a 100-μm nozzle at 20 psi. Cells were sorted into 300 μl of MACS buffer for bulk RNA-seq or 300 μl of 2% BSA in PBS for single-cell RNA-seq. Sample processing was performed in BSL-2 facility using BSL-3 practices. Analysis of the flow cytometry data was performed using FlowJo 10.6.2. using uniform sequential gating strategy (Extended Data Fig. 2) reviewed by three investigators (SS, BDS, AVM). Immune populations were defined using canonical markers as shown in Extended Data Fig. 244, 49. Alveolar macrophages were defined by the expression of CD206 (mannose receptor). We further subdivided alveolar macrophages into CD206lo alveolar macrophages, which represent differentiating monocyte-derived alveolar macrophages, and CD206hi alveolar macrophages, which include both tissue-resident alveolar macrophages, present in the lung prior to the onset of pneumonia, and mature monocyte-derived alveolar macrophages. Since CD206hi alveolar macrophages can be found in BAL fluid from both healthy volunteers and patients across different types of pneumonia, and the presence of CD206lo alveolar macrophages varies across the patients with pneumonia4951, we focused our transcriptomic analysis on CD206hi alveolar macrophages. Abundance of specific populations in individual BAL fluid samples can be found in Supplemental Data File 9.

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



Q&A
请登录并在线提交您的问题
您的问题将发布在Bio-101网站上。我们会将您的问题发送给本研究方案的作者和具有相关研究经验的Bio-protocol成员。我们将通过您的Bio-protocol帐户绑定邮箱进行消息通知。