This protocol has been standardized for cellular specimens, allowing the comparison of experiments running at different synchrotron locations. The main advantage of this technique is a simple and straightforward workflow that allows a fast and reliable preservation of the chemical integration of the sample. Individuals new to this technique may struggle with the frozen iterated cells pelleting and the fast loading of the cellular pellet into the cryosample order.
A precise and quick unlink of a samples in cryogenic temperature is critical. As such, a visual demonstration is a essential to understand how to perform the technique. To prepare human prostate and ovarian cancer cell line cell pellets for selenium speciation, seed the appropriate number of cells from each cell line into three T-75 flasks per condition in the appropriate cell culture medium in a laminar flow hood and place the flasks into 37 degree Celsius and 5%carbon dioxide cell culture incubator until the cells are 80%confluent.
To expose the cancer cells to selenium treatment, first sonicate freshly prepared nanoparticle stock solutions in an ultrasound water bath for 30 minutes at room temperature before serially diluting the selenium nanoparticle solution in complete cell culture medium to the appropriate working concentrations. In the laminar flow hood, gently wash the cells two times with five milliliters of 37 degrees Celsius PBS per wash and use a sterile 25 milliliter pipette to carefully add 15 milliliters of the selenium treatment of interest to the bottom of the flask. Close the lids without completely sealing the flask and place the flasks horizontally in the cell culture incubator for 24 hours.
To prepare the cell pellets after washing and replenishing the culture medium, use one cell scraper per flask to gently detach the cells. Use medium to flush any cells stuck to the flask into the supernatant and collect the dissociated cells by centrifugation. Wash the pellets two times in five milliliters of PBS per tube per wash to remove all remaining traces of the treatment and resuspend the cells in one milliliter of PBS.
Transfer the cells into a 1.5 milliliter polypropylene tube and collect the cells with another centrifugation. Then use a 200 microliter pipette to gently remove all of the supernatant and plunge the bottom part of each 1.5 milliliter tube into liquid nitrogen to the level of the cell pellet. Immediately after freezing, transfer the tubes into a liquid nitrogen dewar for long-term storage.
For high resolution x-ray absorption spectroscopy, optimize all of the crystals from the crystal analyzer spectrometer in Bragg conditions with respect to the fluorescence line energy of interest and use a reference for which the energy position of the absorption edge is known to calibrate the incident monochromatic beam energy. Transfer the sample holder to a liquid helium cryostat for biological samples and set the cryostat to 10 degrees Kelvin, then close the experimental hutch following the synchrotron safety rules, and begin the analysis. Here, representative high resolution x-ray absorption spectroscopy spectra of the selenium in the initial state and in cells incubated to nutritive medium demonstrate that the selenium in the initial selenium nanoparticles was present as both selenium zero and cellulite-like forms.
After interactions with the PC3 cells, however, the selenium was mainly present in the cells as selenium zero, demonstrating a change of selenium species within the cells. For iron, high-resolution x-ray absorption spectroscopy reference spectra exhibit distinct edge positions depending on the iron oxidation state, with reduced species of iron shifted to low energy values. Two successive spectra collected on the same position of a diatom pellet were similar, indicating that the beam damage was limited between two acquisitions when using a helium cryostat at 10 Kelvin.
Furthermore, spectra from different positions of the diatom pellet were identical, demonstrating that the sample pellet was homogenous and that the spectra could be averaged to obtain a better signal-to-noise ratio spectrum. The most critical steps are the preparation of the nanoparticle diltuion, pelleting at cryogenic temperature, and the preparation of the standard reference samples. This procedure can be applied to other bulk analytical techniques, such as ICP-MS or HPLC-ICP-MS or other non-synchrotron spectroscopic techniques able to be performed form at cryogenic temperature.
Other scientific questions can be explored using this procedure, particularly in the fields of biogeology, of biochemistry, and environmental sciences or toxicology research. Selenium compounds and selenium nanoparticles can be as arduous to your health and should be manipulated in a dedicated chemical hood using gloves, glasses, and a face mask.
