Immunology and Infection
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评估对流感嗜血杆菌的呼吸道免疫应答
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Summary June 29th, 2021
Please note that all translations are automatically generated.
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流感嗜血杆菌 诱发呼吸道炎症。本文将重点介绍如何使用流式细胞术和共聚焦显微镜来确定吞噬细胞和淋巴细胞对该细菌的免疫反应。
Transcript
流感嗜血杆菌是各种慢性肺部疾病（包括COPD和肺炎）炎症的主要原因。该协议描述了评估嗜血杆菌对肺部炎症影响的方法。该技术的优点是可以全面评估先天性和适应性免疫反应。
将 450 微升外周血与 50 微升标记为流感嗜血杆菌的活化碘化丙啶在 5 毫升管中在 37 摄氏度的水浴中孵育 20 分钟。从水浴中取出样品，加入五微升DHR并涡旋10秒钟。然后将其放回水浴中再放置 10 分钟。
从水浴中取出样品后，用五毫升0.8%氯化铵溶液裂解红细胞，并如文本中所述在流式细胞仪上分析样品。将外周血样本分成等分试样进行对照和抗原刺激。向两个样品添加共刺激抗体。
然后将非典型流感嗜血杆菌添加到抗原样品中，并在37摄氏度和5%二氧化碳下孵育一小时。接下来，将高尔基体封闭剂布雷菲尔丁A添加到样品中，并再孵育五个小时。如前所述裂解红细胞后，使用 500 微升 1% 至 2% 的副甲醛固定白细胞一小时。
使用血细胞计数器计数细胞，然后用100微升0.1%皂苷透化100万个细胞15分钟。接下来，用适当的荧光标记抗体孵育细胞。洗涤细胞，然后使用流式细胞仪进行分析。
通过获取相关的淋巴细胞群来确定抗原反应细胞的比例。对未受刺激的细胞进行背景染色，以分析所有要分析的细胞因子。将大约 20 到 40 克的肺叶切除术样本切成三到五立方毫米的部分。
将它们放入无菌的50微升腔室中，并使用适当的解聚器机械地碎片组织。组织解聚后，如图所示裂解红细胞，并将细胞重悬于无菌RPMI中。然后通过100微米无菌尼龙网过滤细胞，并使用台盼蓝排除方法计数活细胞。
对于感染测定，将肺细胞重悬于RPMI中，最终浓度为每管每毫升400万个细胞。然后以每个细胞100个细菌的MOI感染细胞。松开盖子旋转半圈，以允许气体在管中转移。
将细胞放入试管旋转器中，并在 37 摄氏度下孵育它们，同时以 12 RPM 旋转。刺激后一小时，加入格雷菲尔丁A以防止细胞因子的细胞外输出，并将细胞悬液再孵育16至22小时。第二天，用含有1%牛血清白蛋白和0.01%叠氮化钠的500微升PBS洗涤细胞悬液。
接下来，对细胞悬液染色特定的人淋巴细胞表面标志物一小时。然后用PBS洗涤细胞并如前所述对其进行固定和透化。接下来，用细胞内细胞因子染色抗体孵育细胞一小时。
然后洗涤细胞并将其重悬于100微升PBS中，然后在流式细胞仪上采集数据。通过金属蛋白酶的作用测量蛋白水解，将荧光素标记的明胶底物直接添加到肺组织切片载玻片上。将载玻片水平放置，并在37摄氏度的避光加湿室中孵育一小时。
为了制备阴性对照，向切片中加入一个不含荧光明胶的反应缓冲液以进行进一步分析。使用向前和侧面分散的外周血单核细胞进行门控，以定义吞噬细胞群。吞噬细胞群通过CD14表达进一步定义以标记单核细胞。
通过氧化DHR 123进行ROS测量以产生荧光。将受激发样品的中位数荧光与对照进行比较。使用流式细胞术测量淋巴细胞产生的细胞内细胞因子。
首先分析细胞白细胞标志物CD45的表达，然后分析CD三和CD四，CD八。将CD三、CD四阳性细胞评估对照和刺激样品中细胞内细胞因子的产生情况。在未固定的肺组织切片中通过NC两个酶谱测量蛋白酶活性。
荧光染色表明存在MMP活性，MMP活性也与染色质的表达共定位。向肺组织样品中添加抗体需要浓缩，细胞染色需要在初步实验中进行优化。肺组织样品的上清液可以使用ELISA等技术进一步分析其他炎症介质，这些技术可用于评估潜在疗法对肺部炎症的影响。
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