This protocol is complementary to in vivo imaging. It allows cell type and cellular level quantification of radiotracers, which is a different scale from the one obtained in vivo. The main advantage of this technique is its sensitivity and the cellular resolution given by the use of radioligands and the cell sorting.
Turn on the camera and load the operating software. Click on the Home XYZ stage button to perform homing. Set up the experiment composed of one 10-minute scan acquisition.
Set the Step mode to fine and the Acquisition mode to the Listmode to record the entire emission spectrum. Set up the bed and ensure that the warming system, breathing sensor, and anesthesia are functional and secure. Then, place a phantom where the animal's head will be positioned.
Set the scan area by sliding the cursors for the three dimensions with the help of the three images in the bottom part of the screen. Ensure that the scan volume of the phantom and the animal is the same. Start the phantom scan for subsequent calibration with the previously established parameters.
Ensure to work in an appropriate environment, authorized for experiments involving radioactivity. Incubate 100 micrograms of tributylin precursor in 100 microliters of acetic acid with sodium iodide and five microliters of 37%paracetic acid at 70 degrees Celsius for 20 minutes in a thermocycler positioned in a glove box. Dilute the reaction using 50%acetonitrile in water to reach a volume of 500 microliters.
Inject the 500 microliters of the diluted reaction onto a reverse-phase column. Isolate the CLINDE with a linear-gradient HPLC run from 5 to 95%ACN in seven-millimolar phosphoric acid for 10 minutes. Dilute the isolate in water to attain a final volume of 10 milliliters and then inject the diluted reaction onto a concentration column.
Elute the CLINDE from the column with 300 microliters of absolute ethanol and then evaporate the ethanol by incubating it in a vacuum centrifuge at room temperature for 40 minutes. Dilute the residue containing the CLINDE in 300 microliters of saline to create a stock solution. After measuring its radioactivity, dilute the stock solution in saline to obtain a solution of 0.037 megabecquerel in 500 microliters.
Establish the calibration curves with cold reference compounds and resolve the linear regression equation:Y equal to AX plus B to obtain A and B coefficients. X represents the mass of cold compound, and Y represents the area under the curves. Check the area under the curve of the radioligand, measuring ultraviolet absorbance at 450 nanometers based on the calibration curve and the area under the curve of the radioligand.
Estimate the mass and measure the activity of the radioligand. Ensure that the specific activity is greater than 1, 000 gigabecquerel per micromole. Click on the Update image button to update the animal position.
Set the scan area by sliding the cursors with the help of the three images in the bottom part of the screen for the three dimensions. Set up the experiment on a 60-minute scan constituting 60 frames of one minute. Reuse all other parameters set up initially.
Inject 500 microliters of the radioactive radiotracer and then flush the tube with 300 microliters of sterile 0.9%sodium chloride. Simultaneously, click on Start acquisition to start the scan. Open the scan reconstruction software and then open the dataset, looking for the file name parameters file created in the folder of the scan.
Select the isotope of interest. Note that the list mode parameter allows multiple isotope selection at this step. Set the different reconstruction parameters like the voxel size, number of subsets, and iterations as described in the manuscript.
Select the output format as NIFTI and then select Start SPECT reconstruction. Use a flat metal spatula and a razor blade to dissect the regions of interest of the brain. Place the tissues in a two-milliliter centrifuge tube and weigh the tissue obtained.
Put the samples into a two-milliliter centrifuge tube with one milliliter of calcium-and magnesium-free HBSS and then centrifuge at 300 times G for two minutes at room temperature and remove the supernatant without disturbing the pellet. Add 1, 900 microliters of enzyme mix one and incubate it for 15 minutes at 37 degrees Celsius while agitating the tubes by inversion every five minutes. Add 30 microliters of enzyme mix two.
Agitate gently with a 1, 000 microliter pipette back and forth 30 times. Then, incubate the mixture for 15 minutes at 37 degrees Celsius while agitating the tubes by inversion every five minutes. Mix gently back and forth with a 200-microliter pipette and then a 10-microliter pipette before incubating the mixture for 10 minutes at 37 degrees Celsius to dissociate the tissues.
Filter the cells with a 70-micrometer cell strainer and add 10 milliliters of calcium-and magnesium-free HBSS. Centrifuge at 300 times G for 10 minutes at room temperature and remove the supernatant without disturbing the pellet. For myelin depletion, resuspend the pellet with 400 microliters of myelin removal buffer and then add 100 microliters of myelin removal beads before incubating for 15 minutes at four degrees Celsius.
Add five milliliters of myelin removal buffer and centrifuge 300 times G for 10 minutes at room temperature. Add 500 microliters of myelin removal buffer and place the suspended pellets into the magnetic field column. Wash the column with one milliliter of myelin removal buffer four times.
Centrifuge at 300 times G for two minutes at room temperature and remove the supernatant without disturbing the pellet. Vortex briefly to dissociate the cells and add five microliters of Fc Block CD32. Add 100 microliters of the mix of primary antibodies of interest and incubate for 20 minutes at four degrees Celsius.
Centrifuge at 350 times G for five minutes at four degrees Celsius and remove the supernatant without disturbing the pellet. Blot the tubes upside down on soft paper. Vortex the tube briefly, then add 100 microliters of the secondary antibody mix and incubate for 15 minutes at four degrees Celsius.
Add one milliliter of myelin removal buffer and repeat the centrifugation. Discard the supernatant and blot the tubes upside down on soft paper. Resuspend the cells in 250 microliters of sterile PBS and proceed directly to cell sorting.
The in vivo SPECT scan of wild-type rats showed higher binding of CLINDE in the site of the lipopolysaccharide injection than in the contra-lateral region of the brain. The ex vivo samples that underwent fluorescence activated cell sorting radioligand-treated tissue, or FACS-RTT, revealed the presence of a higher number of CLINDE binding sites only in microglia, showing that the cellular origin of the CLINDE signal in the ipsilateral side of the brain was microglia. The increase in translocator protein, or TSPO binding, at 12 months in TgF344-AD rats was restricted to astrocytes.
In 24-month-old rats, the increase in TSPO binding was observed due to both astrocytic and microglial alterations. In older TgF344-AD rats, striatal astrocytes displayed a decreased 5HT2AR density when compared with wild-type. A cortical overexpression of TSPO was observed in both astrocytes and microglia of AD subjects compared with age-matched controls when FACS-RTT was performed on human AD postmortem samples.
When working with radioactivity, follow the instruction of radio-protection and wear appropriate personal protection equipment. Then, after the procedure, make sure to decontaminate the working environment. Protein quantification and gene expression analysis can easily be done after this procedure on isolated cell populations.
