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This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.
The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.
The study of the cellular biotransformations of essential or toxic elements requires speciation techniques with high sensitivity and should minimize sample preparation steps that are often prone to modification of chemical species.
Physiological elements such as selenium and iron are known to be particularly difficult to speciate due to their complex chemistry, various stabilities of the selenium or iron species, and their low concentration in the ppm (mg/kg) or even sub-ppm range. Thus, the study of the speciation of these elements by XAS can be extremely challenging. Synchrotron XAS and especially high energy resolution fluorescence detected XAS (HERFD-XAS), which allows a very low signal-to-background ratio1, are available at synchrotron sources to speciate highly diluted elements in complex biological matrices2,3. Conventional fluorescence-XAS measurements can be performed using an energy resolved solid state detector (SSD) with an energy bandwidth ~150–250 eV, on the CRG-FAME beamline at the European Synchrotron Radiation Facility (ESRF)4, while HERFD-XAS measurements need a crystal analyzer spectrometer (CAS), with an energy bandwidth ~1–3 eV, on the CRG-FAME-UHD beamline at the ESRF2. Fluorescence photons are discriminated with respect to their energy with electronic or optical processes respectively.
The sample cryo-preparation is essential to preserve structures and maintain compositional chemical integrity, thus allowing analysis close to the biological native state5. Moreover, analyses performed at cryogenic temperatures as low as 10 K using liquid helium cryogenic cooling (LN2), allow radiation damage to slow down and preserve elemental speciation for XAS. Although some reviews on XAS techniques applied to biological samples report the necessity to prepare and analyze samples in cryogenic conditions (e.g., Sarret et al.6, Porcaro et al.7), none of them clearly describes the related detailed protocol. In this publication, a method for cryo-preparation of cancer cells and plankton microorganisms is described for HERFD-XAS speciation of Se8 and Fe9 at cryogenic temperature.
Good practice for sample preparation and environment during state-of-the-art XAS spectroscopy measurements require 1) a setup; 2) an analysis procedure that limits the effects of radiation damage as much as possible; and 3) a sample (or model compound reference) as homogeneous as possible with respect to the X-ray photons beam size. The first item is taken into account by performing the acquisition at a low temperature, using a liquid helium cryostat. The second item is dealt with by performing each acquisition on a fresh area of the sample by moving it with respect to the beam. Finally, considering the third condition, samples (pellets) and references (powders) are conditioned in pressed bulk pellets in order to limit porosities and inhomogeneities as much as possible, and to avoid roughness with respect to the beam size on the X-ray probed sample surface. We explain how the protocol deals with all of these points.
We used human prostate cell line PC-3 (high metastatic potential) and ovarian cell line OVCAR-3 (which accounts for up to 70% of all ovarian cancer cases) to investigate the antiproliferative properties towards cancer cells of selenium nanoparticles (Se-NPs), and Phaeodactylum tricornutum diatom as a model species to investigate iron sequestration in phytoplankton.
1. Preparation of the human PC-3 and OVCAR-3 cancer cell pellets for selenium speciation
NOTE: The following protocol is adapted from Weekley et al.10. All steps have to be carried out under a cell culture hood under biosafety level 2 conditions and restrictions, using aseptic techniques.
2. Preparation of the plankton cells for iron speciation
NOTE: Synthetic seawater used for this protocol was prepared by adding ultrapure water sea salts, morpholino propanesulfonic acid, ammonium nitrate, sodium nitrate, sodium metasilicate pentahydrate, sodium phosphate monobasic, vitamin stock, trace metal stock, antibiotic stock, and HEPES buffer at pH = 7.9. The concentrations of each component are indicated in the Table of Materials. Details on the culture can be found in Sutak et al.11
3. Reference compound preparation and measurement
NOTE: Reference compounds (solid or liquid) representative of the species and expected to be found in the biological system must be prepared and analyzed by XAS for comparison with experimental biological samples. Reference spectra can be also found in databases12,13,14 and can be used provided that the measurement conditions (e.g., spectral resolution) were similar to the experimental samples.
4. HERFD-XAS: measuring procedure
The main aims of these preparations were to investigate the interaction between selenium nanoparticles (Se-NPs) and cancer cells, and iron binding and sequestration in phytoplankton.
HERFD-XANES spectra of the selenium in the initial state (BSA Se-NPs) and in cells incubated in nutritive medium (BSA Se-NPs after 24 h incubation) are shown in Figure 10. Results showed that selenium in the initial Se-NPs was present as both Se(0) and selenite-like forms, ...
This protocol was used to study the chemical form of selenium and iron in biological samples by X-ray absorption spectroscopy. It focuses on the cryo-preparation and storage of biological samples and references compounds, as well as on the HERFD-XAS measurements.
Cryo-preparation and storage
The cryo-preparation of the bulk biological sample pellets allows preservation of the chemical integrity of the species present in the samples. This is crucial, because speciation ch...
The authors have no conflicts of interest.
We are grateful for financial contributions to the beamline development by CEMHTI (Orleans, France, ANR-13-BS08-0012-01) and Labex OSUG@2020 (Grenoble, France, ANR-10-LABX-0056). The FAME-UHD project is financially supported by the French "grand emprunt" EquipEx (EcoX, ANR-10-EQPX-27-01), the CEA-CNRS CRG consortium and the INSU CNRS institute. We are grateful of all the contributions during the experiments especially all the persons working on BM30B and BM16. The authors acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation beamtime. We also acknowledge PHYTOMET ANR project for financial support (ANR-16-CE01-0008) and SEDMAC project for financial support (INCA-Plan cancer-ASC16019CS).
Name | Company | Catalog Number | Comments |
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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