Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution X-Ray Absorption Spectroscopy

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.

1. Count the cells using a Malassez cell counting chamber. Seed 150,000–200,000 cells per flask for the PC-3 cell line and 300,000 cells for the OVCAR-3 cell line.
2. Seed cells in T-75 flasks (three flasks per condition in order to have triplicates) in the adequate cell culture media (Table 1) under the laminar flow hood.
3. Leave the cell cultures to grow in an incubator at 37 °C and a humidified atmosphere with 5% CO₂ until they reach 80% confluence. Usually, PC-3 prostate cancer cell lines double after 24 h while OVCAR-3 ovarian cancer cell lines take 72 h. The flasks must be left in the incubator.
4. Meanwhile, dilute the Se-NPs used for treatment to a final concentration that corresponds to the IC20 (inhibitory concentration to achieve 20% cell death) that has been predetermined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for cytotoxicity assessment. These concentrations are specific to each Se-NPs type but also specific to the studied cell line.
   1. Place the nanoparticle stock solution (2 mg/mL of bovine serum albumin [BSA] or chitosan coated Se-NPs) in an ultrasound water bath for 30 min at room temperature (RT).
   2. Dilute the Se-NPs solution by serial dilutions in complete cell culture medium to reach working concentrations. Between each dilution, vortex for 1 min.
5. Prepare a selenium positive control with diluted selenium salt for treatment.
   1. Prepare an aqueous sodium selenite stock solution (1 mg/mL) in a 15 mL polypropylene tube. Mix 1 mg of sodium selenite powder with 1 mL of ultrapure water.
   2. Vortex the stock solution for 1 min.
   3. Dilute the sodium selenite stock solution by serial dilutions in complete cell culture medium to reach working concentrations.
      NOTE: Do not forget to pipet several times before each dilution in order to homogenize the solution.
6. Expose the cancer cells to selenium treatments.
   NOTE: This part of the protocol has to be repeated for each nanoparticle (NP) tested. Here, the NPs are of two types, BSA and chitosan coated Se-NPs, plus the controls. The sodium selenite solution is the positive control and no treatment is the negative control.
   1. Open the three T-75 flasks containing the cells in the laminar flow hood.
   2. Remove the medium and wash the cells gently 2x with 5 mL of warmed (37 °C) phosphate buffered saline (PBS).
   3. Gently, on the bottom-side of the flask and not directly on the cell layer, add 15 mL of the treatment in each flask using an automatic pipette equipped with 25 mL sterile plastic pipettes. Place the lids on the flasks. Do not close the lids tightly.
      NOTE: As described in steps 1.3 and 1.4, treatment solutions must have been resuspended beforehand to be homogenized.
   4. Leave the three flasks horizontally in an incubator at 37 °C and an atmosphere humidified with 5% CO₂ for 24 h.
7. Preparation of the cell pellets
   1. Gently wash the cells 2x with 5 mL of warmed PBS (37 °C) directly in the T75 flasks.
   2. Add 6 mL of warmed cell culture medium (37 °C) on the cell layer in the T-75 flask using a pipette boy equipped with 10 mL sterile plastic pipettes.
   3. Collect the cells by gentle scraping using a cell scraper. Use one cell scraper per flask.
   4. Using a pipette boy and a 10 mL pipette, aspirate the liquid and flush back the cell/medium on the flask surface in order to collect all the cells. Repeat this step several times to dissociate and individualize the cells. Collect the medium containing the cells in a 15 mL polypropylene tube.
   5. Spin down at 250 x g for 5 min at RT. Aspirate the supernatant.
   6. Rinse the cells by resuspending them in 5 mL of PBS.
   7. Spin down at 250 x g for 5 min at RT. Aspirate the supernatant and repeat steps 1.6.5 and 1.6.6 2x to get rid of all remaining traces of the treatment.
   8. Resuspend the cells in 1 mL of PBS and transfer them into a 1.5 mL polypropylene tube. Finally, spin them down at 250 x g for 5 min at RT. Figure 1 represents the cell pellet obtained after centrifuging and discarding the supernatant.
   9. Gently remove all the PBS supernatant using a 200 µL pipette.
      NOTE: Be careful to not touch and damage the cell pellet. Ideally, the pellet should be 2–3 mm high or thick, with a diameter of 3–6 mm. For the PC-3 cell line, 9 x 10⁶–10 x 10⁶ cells are required for the assay. For the OVCAR-3 cell line, 8 x 10⁶–9 x 10⁶ cells are required (Figure 1). Some cells will likely be lost during the subsequent buffer rinsing steps. The higher the cell number, the better the pellet will be.
8. Flash-freezing of the cell pellets
   1. Plunge the bottom part of the 1.5 mL tube containing the pellet in liquid nitrogen (LN₂) to the level of the cell pellet.
   2. In a glove box fully purged with an inert atmosphere such as nitrogen gas, collect the cell pellet by gently tapping the 1.5 mL tube on the flat surface of a LN₂-cooled copper block (Figure 2). The pellet is then collected without cell loss.
      NOTE: Use cryoprotection equipment, lab coat, closed shoes, cryo-gloves, and a face shield or safety goggles for LN₂ handling.
   3. If needed, a LN₂ cooled needle can be used to help to take out the pellet.
      NOTE: The resulting frozen cell pellet can fragment. These steps must be performed in the space of a few minutes and rely on the researcher's dexterity and speed.
   4. The frozen pellet dimensions must fit the cryostat sample holder. Using the equipment described, if the cell pellet is larger than 2 mm in height and slightly smaller than the hole of the sample holder used for XAS, the sample can be used directly. If not, or if a flat surface for the analysis is desired, a bulk cylindrical pellet must be prepared, still keeping the pellet in its frozen state. If the cell pellet is fragmented the following steps can be used ideally in a glove box fully purged with an inert atmosphere.
9. Preparation of the bulk frozen cylindrical pellets
   1. Immerse all the parts of a manual hydraulic press that will be in contact with the sample in LN₂ (Figure 3A; 3- or 5-mm diameter pellet dies, mold, piston/wire pulling).
   2. Transfer the rapidly frozen pellet fragments into the mold loaded with the appropriate pellet dies (Figure 3B).
   3. Quick pelletize with the hydraulic press. Use of 1.5 tons for a 5 mm diameter pellet or 0.5 tons for a 3 mm diameter pellet is sufficient (Figure 3C).
      NOTE: After each pellet preparation with the hydraulic press, all the materials need to be thawed and dried properly using nitrogen gas to avoid any problems with the remaining frost and humidity.
   4. Collect the bulk pellet of frozen cells quickly and place it in an adequate cryotube for storage.
   5. Store it back into LN₂ tank for long-term storage.
   6. Transfer and mount the cryo-pellets for analysis. The pellet stored in a LN₂ cooled cryotube is transferred to a 77 K precooled cryostat sample holder in liquid N₂ (Figure 4, as used for a CRG-FAME-UHD beamline). This step should be performed as quickly as possible but can take a few minutes. Then transfer the sample holder to the cryostat and maintain it at 10 K during the whole XAS analysis.
      NOTE: Steps 1.9.3 and 1.9.6 are difficult because they need to be done quickly in order to avoid any frost formation that will block the piston and the mold. An alternative is to perform these steps under a plastic tent fully purged with nitrogen gas. The LN₂-cool copper mass allows the pellet to be kept frozen.

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

1. Centrifuge a P. tricornutum diatom culture in a 50 mL polypropylene tube at 1,100 x g for 6 min and transfer the pellet into a flask with fresh medium.
2. Let the culture grow in synthetic seawater in a growth chamber for 24 h.
3. Centrifuge the plankton culture for 6 min at 1,100 x g and transfer the pellet into fresh medium containing Fe-citrate (1 µM in the final culture). Incubate for 72 h.
4. Prepare the plankton cells
   1. Centrifuge the cells for 3 min at 1,100 x g and rinse with medium without iron.
   2. Transfer the pellet into a 1.5 mL polypropylene tube, wash with ultrapure water, and centrifuge 2x for 3 min at 1,100 x g. Remove the supernatant.
   3. Plunge the bottom part of the 1.5 mL polypropylene tube containing the pellet in LN₂ as described in step 1.8.
   4. Transfer the frozen cells in a LN₂ frozen molder and quickly press using a XRF pellet press (Figure 3 and Figure 7) as described in step 1.9.
   5. Remove the pellet and store it in a LN₂ tank for long-term storage.
   6. Follow step 1.9.6 (see Figure 4 for the transfer into the cryostat sample holder).

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 databases^12,13,14 and can be used provided that the measurement conditions (e.g., spectral resolution) were similar to the experimental samples.

1. For references in aqueous solution, weigh initial compounds in powder or liquid form under anaerobic condition or inert atmosphere. Prepare redox-sensitive references in an inert atmosphere (i.e., in an anaerobic glove box or in a Schlenk line, see Figure 5 and Figure 6) to avoid any oxido-reductive reaction and to preserve the chemical integrity of the compound, which can be highly reactive and unstable in ambient air). Here, all the selenium references were prepared using a Schlenk line and degasified ultrapure water (Figure 6). Liquid Fe^III-malate and ferritine were prepared at ambient air.
2. Mix with appropriate solution in order to reach a 1% wt final concentration of the studied element (i.e., selenium or iron) for collection in fluorescence mode.
   NOTE: Ultrapure water is the most frequently used solution to prepare XAS references. Addition of glycerol (15%–20%) is required for liquids measured using standard XAS fluorescence to prevent artifacts arising from X-ray diffraction by ice crystals and preserved quality of the XAS signal collected with the solid-state detector. However, for HERFD-XAS, glycerol is not mandatory. Using a crystal analyzer spectrometer instead of a solid-state detector, the energy is selected with an extremely high-resolution and the diffraction induced by ice crystals does not impact the data collection of the studied element¹.
3. Preserve and store the prepared solution in a sealed Schlenk balloon (Figure 5) or in a 1.5 mL polypropylene tube until measurement in the same conditions as the samples.
4. For solid references, weigh selenium or iron model compound powders at ambient air or in a glove box if the species are redox-sensitive. Here, solid Fe^II references (Fe^II-acetate and vivianite) were prepared in a gaseous N₂ glove box while Fe^III (Fe(OH)₃, and Fe^III-phosphate) were prepared at ambient air.
5. Weigh high purity boron nitride powder and mix with the model compounds powders in order to reach a 1% wt final concentration of the studied element (Figure 7A).
6. Grind to a fine powder using a mortar for at least 15 min.
7. Place the powder mixture in the molder to prepare pellets with the hydraulic press (Figure 7B, similar to Figure 3A for the sample pellet).
8. Close the molder with the piston (Figure 7C, similar to Figure 3B for the sample pellet).
9. Press to obtain the bulk pellet (Figure 7D, similar to Figure 3C for the sample pellet).
10. Store the prepared bulk pellets in the glove box until they are analyzed in the same conditions as the samples.
    NOTE: The bulk pellet sometimes sticks to the pistons, depending on the powder compacity, for example. Removing it softly without breaking it then can be difficult. In that case, the following procedure using polyimide (e.g., Kapton) disks has to be used.
11. Put a drop of ethanol on each side of the pistons that will be in contact with the powder (Figure 8A).
12. Prepare two polyimide disks with a diameter slightly smaller than the pistons. The polyimide thickness needs to be 10–25 µm (thinner will be difficult to manipulate, thicker will absorb too much of the X-ray photons during analysis).
13. Put the disks on the pistons. They will stick by capillary action (Figure 8B).
14. Mount the molder with the piston, add the powder, and put in the second piston (Figure 8C).
15. Press (see step 3.11) and remove the compressed bulk pellet from the pistons.
    NOTE: The solid references can be mounted on the cryostat-specific sample holder like the samples (see step 1.8.6). The liquid references can be mounted on the cryostat-specific sample holder. See Figure 9A for a CRG-FAME cryostat sample holder. The same procedure can be applied using a CRG-FAME-UHD sample holder, shown in Figure 4D, using the following procedure.
16. Mounting of liquid reference on the cryostat sample holder.
    1. Set the polyimide (e.g., Kapton) tape (25 µm thick) in order to seal one side of the hole on the sample holder at RT (Figure 9B).
    2. Using a syringe, fill the cavity slowly with the solution until it reaches the top. Avoid bubbles. The reservoir must be filled (Figure 9C, left).
    3. Seal the other side of the cavity with the tape (Figure 9C, right).
    4. Plunge the sealed sample holder into LN₂ (Figure 9D, T₀–T₃).
    5. A thermic reaction induces LN₂ bubbling. Wait until bubbling stops, signaling the end of the reaction (Figure 9D, T₃–T₅).
    6. Transfer the sample holder at 77 K into the cryostat of the beamline before starting data collection (Figure 9D, T₆) or storing in the LN₂ Dewar.
       NOTE: The frozen solution is well spread in the cavity and forms a uniform and homogeneous pellet (Figure 9D, T₇).

4. HERFD-XAS: measuring procedure

1. Optimize all the crystals from the CAS in Bragg conditions with respect to the fluorescence line energy of interest. This procedure can be done with a concentrated reference compound.
2. Calibrate the incident monochromatic beam energy using a reference for which the energy position of the absorption edge is known (typically a pure metallic foil). This reference can be positioned in a second step after the sample, in double transmission mode, in order to be able to check the calibration for each obtained spectra and eventually align them posttreatment.
3. Position the sample in a liquid helium cryostat for biological samples following the appropriate procedure described in step 1.9.6.
4. Close the experimental hutch following the synchrotron safety rules.
5. Perform the XAS acquisition on an energy range that covers the selected absorption edge.
6. After each spectral acquisition, move the sample at least twice the beam size in the direction of the motion before starting a new acquisition.
   NOTE: In this case, measurements were performed using Ge(440) crystals to select the Fe K_a1 line and using Ge(844) crystals to select the Se K_a1 line. The beam size on the sample was 200 x 100 µm² (H x V Full Width Half Maximum). The sample motion between each acquisition was 500 µm in both directions, in order to probe structural homogeneity and to analyze a new area free from previous possible radiation damage each time. Individual spectra are compared and merged if they are superimposable within the noise. The measurement is stopped for a given sample when the quality of the merged spectrum is correct. Then data analysis can be performed using any software dedicated to XAS analysis, especially those that allow a linear combination fitting of normalized spectra, for example Athena and Artemis (Demeter group of software)¹⁵, SixPack¹⁶, or Viper¹⁷. Complete and detailed explanations of XAS analysis falls outside the scope of this protocol and can be found in recent papers^18,19, some of them more specifically devoted to biological samples²⁰. Selenium XANES reference spectra are already gathered in the SSHADE spectra database²¹.