A nanovaccine for immune activation and prophylactic protection of atherosclerosis in mouse models

The raw flow cytometry files uploaded contain the activation of bone marrow derived dendritic cells (BMDCs), the proliferation profile after co-culture of BMDCs and T lymphcytes. We also analyzed splenic and lymphatic cells from C56BL/6J and ApoE knockout (KO) mice after vaccination of our nanovaccines (D+P, SP-D1P1, SP-D1+P1, and SP-P).

Methods
For in vitro experiments such as to check the surface expression of different markers, the collected cells were stained with antibodies before staining with Zombie Aqua fixable viability dye  Finally, the cells were resuspended in 200 µL FACS buffer (5 mM EDTA and 1% FBS in PBS) for flow cytometry analysis by BD FACSCantoII or a BD FACSCeleste cell analyzer. Data were analyzed by the FlowJo software (Tree Star) or the FCS Express 7 software (Reachsoft).

For in vivo experiments at 10 weeks and 26 weeks of age, the immunized ApoE-/- mice were anesthetized for blood collection and euthanized as indicated above. The spleens and dLNs were excised, minced, and filtered (70 μm; BD Biosciences) to generate a single-cell suspension and centrifuged at 4 °C at 1800 rpm for 10 min. After lysis of red blood cells, the pelleted splenocytes were resuspended in FACS buffer together with antibodies. The dLNs were extracted and passed through a 70 μm cell strainer, stained with antibodies. The cells were then stained with Zombie Aqua Fixable Viability Kit and resuspended in 200 µL of FACS buffer for flow cytometry analysis by a flow cytometer (BD FACSCantoII). Data were analyzed by the FlowJo software (Tree Star).

本次上传的原始流式细胞术（flow cytometry）文件包含骨髓源树突状细胞（bone marrow derived dendritic cells, BMDCs）的活化情况，以及BMDCs与T淋巴细胞共培养后的增殖谱。本研究还对使用本团队开发的纳米疫苗（D+P、SP-D1P1、SP-D1+P1及SP-P）免疫的C57BL/6J小鼠与载脂蛋白E基因敲除（ApoE knockout, ApoE KO）小鼠的脾脏及淋巴结细胞进行了分析。

实验方法

体外实验：为检测不同标志物的细胞表面表达水平，收集的细胞先经特异性抗体染色，随后使用Zombie Aqua固定活性染料进行染色。最终将细胞重悬于200 μL FACS缓冲液（PBS中含5 mM EDTA与1%胎牛血清（FBS））中，采用BD FACSCantoII或BD FACSCeleste细胞分析仪开展流式细胞术分析。数据通过FlowJo软件（Tree Star公司）或FCS Express 7软件（Reachsoft公司）进行处理。

体内实验：针对10周龄与26周龄的体内实验，对免疫后的ApoE-/-小鼠实施麻醉以采集血液，并按前述方法实施安乐死。摘取脾脏及引流淋巴结（dLNs），将组织剪碎后通过70 μm滤膜（BD Biosciences公司）过滤以制备单细胞悬液，随后于4 ℃下以1800 rpm离心10分钟。红细胞裂解后，将沉淀的脾细胞重悬于FACS缓冲液中并加入特异性抗体进行染色。引流淋巴结组织经摘取后通过70 μm细胞筛过滤，再用特异性抗体染色。随后使用Zombie Aqua固定活性染料对细胞进行染色，并重悬于200 μL FACS缓冲液中，采用流式细胞仪（BD FACSCantoII）完成流式细胞术检测。数据通过FlowJo软件（Tree Star公司）进行分析处理。