Data from: Single-cell analysis identifies conserved features of immune dysfunction in simulated microgravity and spaceflight
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3-dimensional super-resolution microscopy volumes of human PBMCs recorded on a Zeiss LSM980 Airyscan2 laser scanning confocal microscope. Sample preparation and image capture: Live PBMCs were stained with 60 nM MitoTracker Red-CMX-Ros (ThermoFisher, Waltham, MA) either in 6-well plates or in the microgravity chambers for the last 2 hr of the microgravity simulation. At the end of the microgravity simulation cells were immediately fixed by 1:1 mixing the cell suspensions with 2× concentrated fixative (10% Sucrose (w/v) 120 mM KCl, 1% (w/v) glutaraldehyde, 8% (w/v) PFA pH 7.4) and incubated for 15 minutes at room temperature followed by 15 minutes on ice. Fixed cells were washed and stored in PBS until further staining for up to a week at 4 °C. 1 million fixed cells were resuspended in 1 mL of permeabilization solution (0.1% TritonX-100 in PBS) for 5 minutes. After twice washing in PBS, pellets were resuspended in 0.5 mL 1% BSA PBS containing Phalloidin-iFluor-488 (cat# ab176753, Abcam plc., Cambridge, UK) at the manufacturer’s recommended dilution, and were incubated for 90 minutes with gentle agitation. After washing in PBS, cells were stained with Hoechst 33342 (1 µg/mL in PBS) for 10 minutes. The fixed-stained cells were immobilized at 3 × 10<sup>5</sup> cells per well density in glass-bottom 96-well microplates (Greiner Bio-One, Monroe, NC), which were pre-coated with polyethyleneimine (1:15,000 (w/v)) for 16 hours in a 37 °C incubator, and washed twice with PBS. Microplates with the cell suspensions were centrifuged in a swing plate rotor centrifuge (Eppendorf 5810 R) at 400 × <em>g</em> and for 10 min and then fixed on the surface by adding an equal volume of 8% (w/v) PFA for 5 min. Finally, the fixative was replaced with 100 µL of antifade reagent (Vector Prolong Gold (ThermoFisher)). Samples were imaged immediately after this procedure on a Zeiss LSM980 Airyscan2 laser scanning confocal microscope (Carl Zeiss Microscopy, White Plains, NY). Single PBMCs were manually selected for recording based on low-resolution preview scans showing only nuclei. All singlet cells were selected in a small neighborhood to avoid biases. In each microscopy session, 24-40 cells were selected for recording in one well for each condition. This was performed in an interleaved manner, capturing 6-8 cells at a time, and then moving to the next well and then repeating this multiple times using the Experiment Designer module for automation. Super-resolution volumes of (358 × 358 × 70 pixels, 0.035 × 0.035 × 0.13 µm/voxel resolution) were recorded in the above-determined positions using Definite Focus autofocusing. A Plan-Apochromat 63 × 1.40 Oil lens, Airyscan2 SR (super-resolution) mode with optimal sampling and frame switching between 3 fluorescence channels to minimize spectral cross-bleed were used. MitoTracker Red, iFluor488, and Hoechs33342 were excited with 561, 488, and 405 nm solid-state lasers, respectively, using the optimal emission filter for each channel. 3D Airyscan2 processing was performed with standard filtering settings. File naming: Four zip files were deposited named as <Donor#id>.zip, where id goes from 1 to 4. Each zip file contains the following Zeiss Microscopy format image files: <Condition>_<Donor#id>_<Stain#batch>_<Cell>.czi <Condition>: 1G – Control culturing in 6-well plates for 25h uG – simulated microgravity culturing for 25h in NASA Rotating Wall Vessels 1G+TLR – as above, with TLR 7/8 agonist (1 μM R848) uG+TLR– as above, with TLR 7/8 agonist (1 μM R848) 1G+CyD– as above, with cytochalasin D uG+CyD– as above, with cytochalasin D 1G+Q – as above, with quercetin 50µM uG+Q – as above, with quercetin 50µM <Donor#id>: 1-4 indicates biological replicates <Stain#batch>: 1-2 indicates experimental replicates of phalloidin staining and imaging session <Cell>: arbitrary number to distinguish images within the same condition/donor/stain set. See image analysis pipelines used with these data at: https://github.com/gerencserlab/Superresolution-actin-and-mitochondria-analysis
本数据集为在蔡司LSM980 Airyscan2激光扫描共聚焦显微镜下采集的人外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）三维超分辨率显微成像体积。 ### 样本制备与图像采集 将活的人外周血单个核细胞置于6孔板或微重力培养舱中，在微重力模拟的最后2小时，用60 nM的MitoTracker Red-CMX-Ros（赛默飞世尔科技，马萨诸塞州沃尔瑟姆）进行染色。微重力模拟结束后，立即将细胞悬液与2×浓缩固定液（10%蔗糖（质量体积比）、120 mM氯化钾、1%（质量体积比）戊二醛、8%（质量体积比）多聚甲醛（PFA），pH 7.4）按1:1体积比混合，对细胞进行固定：先在室温孵育15分钟，随后置于冰上静置15分钟。固定后的细胞经洗涤后重悬于磷酸盐缓冲液（PBS）中，于4℃储存备用，储存时长不超过一周。 取100万固定后的细胞，重悬于1 mL透膜液（含0.1% Triton X-100的PBS）中孵育5分钟。经PBS洗涤两次后，将细胞沉淀重悬于0.5 mL含1%牛血清白蛋白（BSA）的PBS中，其中加入按厂商推荐浓度稀释的Phalloidin-iFluor-488（货号ab176753，英国剑桥Abcam公司），轻柔振荡孵育90分钟。经PBS洗涤后，用Hoechst 33342（1 μg/mL的PBS溶液）对细胞染色10分钟。 将固定并染色后的细胞以每孔3×10^5个的密度接种于包被有聚乙烯亚胺（质量体积比1:15000）的玻璃底96孔板中：该96孔板需先在37℃培养箱中包被16小时，随后用PBS洗涤两次。将含细胞悬液的96孔板置于水平转子离心机（艾本德5810 R）中，以400×g离心10分钟，随后加入等体积的8%（质量体积比）多聚甲醛，静置5分钟以固定细胞于孔底表面。最后，弃去固定液，每孔加入100 μL抗荧光淬灭剂（赛默飞世尔科技的Vector Prolong Gold）。 完成上述操作后，立即在蔡司LSM980 Airyscan2激光扫描共聚焦显微镜（美国纽约怀特普莱恩斯卡尔蔡司显微镜公司）下对样本进行成像。基于仅显示细胞核的低分辨率预览扫描图像，手动挑选单个PBMC进行成像；为避免偏倚，所有单个细胞均在小邻域范围内选取。每次显微成像批次中，每种培养条件下每孔选取24~40个细胞进行成像。成像采用交错式流程：每次采集6~8个细胞后，移至下一孔，重复该流程多次，全程通过实验设计模块（Experiment Designer）实现自动化操作。 采用Definite Focus自动对焦系统，在上述选定位置采集超分辨率成像体积，参数为：358×358×70像素，体素分辨率0.035×0.035×0.13 μm/体素。成像采用Plan-Apochromat 63×1.40油浸物镜，开启Airyscan2超分辨率（SR）模式，采用最优采样策略，并在3个荧光通道间进行帧切换以最小化光谱串色。MitoTracker Red、iFluor488与Hoechst 33342分别通过561 nm、488 nm与405 nm固态激光器激发，并为每个通道配置最优的发射滤光片。采用标准滤波参数进行3D Airyscan2图像处理。 ### 文件命名规则 共上传4个压缩文件，命名格式为<Donor#id>.zip，其中id取值为1至4。每个压缩文件包含以下蔡司显微镜格式的图像文件：<Condition>_<Donor#id>_<Stain#batch>_<Cell>.czi - <Condition>（培养条件）： 1G：在6孔板中常规培养25小时的对照组 uG：在NASA旋转壁式生物反应器中模拟微重力培养25小时的实验组 1G+TLR：同1G组，同时加入TLR7/8激动剂（1 μM R848） uG+TLR：同uG组，同时加入TLR7/8激动剂（1 μM R848） 1G+CyD：同1G组，同时加入细胞松弛素D（cytochalasin D） uG+CyD：同uG组，同时加入细胞松弛素D 1G+Q：同1G组，同时加入50 μM槲皮素 uG+Q：同uG组，同时加入50 μM槲皮素 - <Donor#id>：取值1-4，代表4个生物学重复样本 - <Stain#batch>：取值1-2，代表鬼笔环肽染色与成像批次的2个实验重复 - <Cell>：任意编号，用于区分同一培养条件、供体、染色批次下的不同图像 可访问以下链接获取针对本数据集的图像分析流程：https://github.com/gerencserlab/Superresolution-actin-and-mitochondria-analysis
