使用高分辨率X射线吸收光谱在低温下制备生物样品以进行形态分析
1University Grenoble Alpes,INSERM UA7, Synchrotron Radiation for Biomedicine (STROBE), 2CNRS, UMR 5254, Université Pau & Pays Adour, E2S UPPA,Institut des Sciences Analytiques et de Physicochimie pour l’Environnement et les Matériaux, 3Institut Jacques Monod, UMR 7592 CNRS,Université Paris Diderot, 4OSUG, UMS 832 CNRS,Université Grenoble Alpes, 5FAME and FAME-UHD beamlines,ESRF, the European Synchrotron, 6ID16A beamline,ESRF, the European Synchrotron
Summary
该协议提出了为基于同步加速器的X射线吸收光谱实验制备生物冷冻样品的详细程序。我们通过癌症和浮游植物细胞方案的示例描述了优化样品制备和冷冻保存所需的所有步骤。该方法提供了样品冷冻制备的通用标准。
Abstract
在研究金属在生物系统中的作用时，用X射线吸收光谱（XAS）研究元素特别有趣。样品制备是一个关键且通常复杂的过程，特别是对于生物样品。虽然X射线形态形成技术被广泛使用，但尚未为该技术的用户传播详细的协议。此外，化学状态修饰是值得关注的，并且建议使用基于冷冻的技术来分析生物样品在其接近天然的水合状态下，以提供细胞或组织化学完整性的最大保存。在这里，我们提出了一种基于冷冻保存样品的细胞制备方案。它在高能分辨率荧光检测的X射线吸收光谱研究中证明了硒在癌细胞中的研究和浮游植物中铁的研究。该协议可与其他生物样品和其他可能被辐照破坏的X射线技术一起使用。
Introduction
对必需或有毒元素的细胞生物转化的研究需要具有高灵敏度的物种形成技术，并应最大限度地减少通常容易改变化学物质的样品制备步骤。
已知硒和铁等生理元素特别难以形成，因为它们具有复杂的化学性质，硒或铁物种的各种稳定性，以及它们在ppm（mg / kg）甚至亚ppm范围内的低浓度。因此，研究XAS对这些元素的形态可能极具挑战性。同步加速器XAS和特别高能分辨率荧光检测的XAS（HERFD-XAS），允许非常低的信背景比1，可在同步加速器源上形成复杂生物基质中高度稀释的元素2，3。传统的荧光XAS测量可以使用能量带宽约为150-250 eV的能量分辨固态探测器（SSD）在欧洲同步辐射设施（ESRF）4的CRG-FAME光束线上进行，而HERFD-XAS测量则需要在ESRF2的CRG-FAME-UHD光束线上使用能量带宽约为1-3 eV的晶体分析仪光谱仪（CAS）.荧光光子分别通过电子或光学过程来区分其能量。
样品冷冻制备对于保存结构和维持成分化学完整性至关重要，因此允许分析接近生物天然状态5。此外，使用液氦低温冷却（LN2）在低至10 K的低温下进行的分析允许辐射损伤减缓并保持XAS的元素形态。虽然一些关于应用于生物样品的XAS技术的综述报告了在低温条件下制备和分析样品的必要性（例如Sarret等人6，Porcaro等人7），但没有一篇明确描述相关的详细方案。在本出版物中，描述了一种用于癌细胞和浮游生物微生物的冷冻制备方法，用于在低温下实现Se8 和Fe9 的HERFD-XAS形态。
在最先进的XAS光谱测量期间，样品制备和环境的良好实践需要1）设置;2）尽可能限制辐射损害影响的分析程序;3）样品（或模型化合物参考）相对于X射线光子光束尺寸尽可能均匀。通过使用液氦低温恒温器在低温下进行采集来考虑第一项。第二项是通过在样品的新区域上执行每次采集来处理的，方法是将其相对于光束移动。最后，考虑到第三个条件，样品（颗粒）和参比物（粉末）在压制的块状颗粒中进行调节，以尽可能限制孔隙率和不均匀性，并避免相对于X射线探测样品表面上的光束尺寸的粗糙度。我们解释了该协议如何处理所有这些要点。
我们利用人前列腺细胞系PC-3（高转移电位）和卵巢细胞系OVCAR-3（占所有卵巢癌病例的70%）来研究硒纳米颗粒（Se-NPs）和 Phaeodactylum tricornutum 硅藻对癌细胞的抗增殖特性，作为研究浮游植物铁螯合的模型物种。
Protocol
1.制备用于硒形态的人类PC-3和OVCAR-3癌细胞颗粒 注：以下协议改编自Weekley等人10。所有步骤都必须在生物安全2级条件和限制下的细胞培养罩下使用无菌技术进行。 使用马拉塞兹细胞计数室对细胞进行计数。PC-3细胞系的每个烧瓶接种150，000-200，000个细胞，OVCAR-3细胞系接种300，000个细胞。 T-75烧瓶中的种子细胞（每种条件下三个烧瓶，以便一式?…
Representative Results
这些制剂的主要目的是研究硒纳米颗粒（Se-NPs）与癌细胞之间的相互作用，以及浮游植物中的铁结合和封存。 在初始状态（BSA Se-NPs）和在营养培养基中孵育的细胞（BSA Se-NPs）中孵育的硒的HERFD-XANES光谱如图 10所示。结果表明， 初始Se-NPs中的硒同时以Se（0） 和亚硒酸盐样形式存在， 而与PC-3细胞相互作用后， 细胞中的硒主要以Se（0）的形式<…
Discussion
该方案用于通过X射线吸收光谱研究生物样品中硒和铁的化学形式。它侧重于生物样品和参比化合物的冷冻制备和储存，以及HERFD-XAS的测量。
冷冻制备和储存
散装生物样品颗粒的冷冻制备可以保留样品中存在的物质的化学完整性。这是至关重要的，因为在使用冷冻干燥或空气干燥制备6时已经观察到形态变化。一旦选择用于测量的光束线配备了…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们感谢CEMHTI（法国奥尔良，ANR-13-BS08-0012-01）和Labex OSUG@2020（法国格勒诺布尔，ANR-10-LABX-0056）为光束线开发提供的财政捐助。FAME-UHD项目由法国“grand emprunt”EquipEx（EcoX，ANR-10-EQPX-27-01），CEA-CNRS CRG财团和INSU CNRS研究所提供财政支持。我们感谢实验期间的所有贡献，特别是所有从事BM30B和BM16工作的人员。作者承认欧洲同步加速器辐射设施提供同步加速器辐射束时间。我们还感谢PHYTOMET ANR项目的财务支持（ANR-16-CE01-0008）和SEDMAC项目的财务支持（INCA-Plan cancer-ASC16019CS）。
Materials
| Ammonium nitrate | Sigma-Aldrich | A3795 | NH4NO3, 2.66 mg/L of milliQ water |
| Anaerobic chamber | Coy Laboratory, USA | equipped with Anaerobic Monitor (CAM-12) | |
| Antibiotic stock | Sigma-Aldrich | A0166 for ampicillin, S9137 for streptomycin sulfate | 1 mL/L of milliQ water (ampicillin sodium and streptomycin sulfate, 100 mg/mL) |
| Boron nitride powder | Sigma-Aldrich | 255475 | |
| Cell counting chamber | Neubauer or Malassez | ||
| Cell scraper | |||
| Dulbecco's Phosphate Buffered Saline (DPBS) | GIBCO | 14190-094 | Without Calcium, Magnesium, Phenol Red |
| Eppendorf tubes | 0.5 mL and 1.5 mL | ||
| Falcon tubes | 15 mL and 50 mL | ||
| Ferric citrate Fe/citrate = 1/20 | Sigma-Aldrich | F3388 | aqueous solution of FeCl3 50 mM and Na-citrate 1M pH 6.5 |
| Fetal Bovine Serum | GIBCO | A31604-02 | Performance Plus, certified One Shot format, US origin |
| Flasks | Sigma-Aldrich | Z707503 | TPP 150 cm2 area |
| Growth chamber | Sanyo | Sanyo MLR-352 | at 20 °C and under a 12:12 light (3,000 lux) dark regime |
| HEPES buffer | Sigma-Aldrich | H4034 | 1 g/L of milliQ water HEPES |
| High grade serous, OVCAR-3 | ATCC, Rockville, MD | HTB-161 | Storage temperature: liquid nitrogen vapor temperature |
| Incubator | Incubator at 37°C, humidified atmosphere with 5% CO2 | ||
| Insulin solution from bovine pancreas | Sigma-Aldrich | I0516 | 10 mg/mL insulin in 25mM HEPES, pH 8.2, BioReagent, sterile-filtered, suitable for cell culture |
| Manual hydraulic press | Specac, USA | ||
| Marine diatom Phaeodactylum tricornutum | Roscoff culture collection | RCC69 | http://roscoff-culture-collection.org/rcc-strain-details/69 |
| Morpholinepropanesulfonic acid | Sigma-Aldrich | M3183 | MOPS, 250 mg/L of milliQ water (pH 7.3) |
| Optical microscope | |||
| PC-3 | ECCAC, Salisbury, UK | 90112714 | Storage temperature: liquid nitrogen vapor temperature |
| Penicillin-Streptomycin | Sigma-Aldrich | P4333 | Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, sterile-filtered, BioReagent, suitable for cell culture |
| Pipette-boy | 25mL-, 10mL-, and 5mL sterile plastic-pipettes | ||
| Plankton culture products, Mf medium: Sea salts | Sigma-Aldrich | S9883 | 40g/L of milliQ water. Composition: Cl- 19.29 g, Na+ 10.78 g, SO42- 2.66 g, Mg2+ 1.32 g, K+ 420 mg, Ca2+ 400 mg, CO32- /HCO3- 200 mg, Sr2+ 8.8 mg, BO2- 5.6 mg, Br- 56 mg, I- 0.24 mg, Li+ 0.3 mg, F- 1 mg |
| Plastic tweezers | Oxford Instrument | AGT 5230 | |
| RPMI MEDIUM 1640 (ATCC Modification) | GIBCO | A10491-01 | Solution with 4.5 g/L D-glucose, 1.5 g/L Sodium Bicarbonate, 110 mg/L (1 mM) Sodium Pyruvate, 2.388 g/L (10 mM) HEPES buffer and 300 mg/L L-glutamine for research use |
| Selenium nanoparticles (Se-NPs), BSA coated, 2 mg/mL | NANOCS Company, USA | Se50-BS-1 | BSA stabilized Se-NPs solution. Average size about 30 nm. Stored at 4°C in the dark, protected from the light. |
| Selenium nanoparticles (Se-NPs), Chitosan coated, 2 mg/mL | NANOCS Company, USA | 11. Se50-CS-1 | Chitosan stabilized Se-NPs solution. Average size about 30 nm. Stored at 4°C in the dark, protected from the light. |
| Sodium metasilicate pentahydrate | Sigma-Aldrich | 71746 | Na2SiO3.5H2O, 22.8 mg/L of milliQ water |
| Sodium nitrate | Sigma-Aldrich | S5022 | NaNO3, 75 mg/L of milliQ water |
| Sodium phosphate monobasic | Sigma-Aldrich | S5011 | NaH2PO4, 15 mg/L of milliQ water |
| T-75 flasks | |||
| Tissue culture hood | |||
| Trace metal stock | Sigma-Aldrich | M5005, Z1001, M1651, C2911, 450243, 451193, 229857 | 1 mL/L of milliQ water (MnCl2.4H2O 200 mg/L, ZnSO4.7H2O 40 mg/L, Na2MoO4.2H2O 20mg/L, CoCl2.6H2O 14 mg/L, Na3VO4.nH2O 10 mg/L, NiCl2 10 mg/L, H2SeO3 10 mg/L) |
| Trypan Blue Solution (0.4%) | GIBCO | 15250061 | |
| Trypsin-EDTA (0.05%), phenol red | GIBCO | 25300-054 | |
| Vitamin stock | Sigma-Aldrich | T1270 for thiamine, B4639 for biotin, V6629 for B12 | 1 mL/L of milliQ water (thiamine HCl 20 mg/L, biotin 1 mg/L, B12 1 mg/L) |
| Water bath 37°C |
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Citar este artículo
Bissardon, C., Isaure, M., Lesuisse, E., Rovezzi, M., Lahera, E., Proux, O., Bohic, S. Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution X-Ray Absorption Spectroscopy. J. Vis. Exp. (183), e60849, doi:10.3791/60849 (2022).