The overall goal of the following experiment is to synthesize an in vivo MRI detectable apoptosis probe. This is achieved by oxidizing MRI, detectable iron oxide nanoparticles or SPIO and conjugating them to a nexin protein. Next apoptosis is induced in adult mouse bone marrow mesenchymal cells that are then incubated with the A-N-X-S-P-I-O probe, which attaches to apoptotic cells.
Finally, MRI is used to image the cell culture dishes in order to evaluate the effectiveness of the probe. Results are obtained that show a signal from the targeted attachment of A-N-X-S-P-I-O onto apoptotic cells based on MRI imaging. The implications of this technique extend toward the therapy and diagnosis of myocardial infarction because early detection will allow for early preventive treatment To conjugate an xin five to SPIO begin by oxidizing SPIO particles in a solution of 0.15 molar sodium per iodate for one hour in the dark at 20 degrees Celsius.
Incubate the oxidized SPIO with purified A NX protein in 0.15 molar sodium bore eight for 12 hours at 20 degrees Celsius to reduce the mixture further under the fume hood. Add one milliliter of sodium boro hydride incubate for one hour. Then quench the solution with one milliliter of 0.15 molar tris HCL to separate the free A NX from the A-N-X-S-P-I-O conjugates.
Apply the reaction to a 75 millimeter centrifugal filter and spin at 14, 000 times G for 15 minutes. Remove from the centrifuge to obtain the supernatant. Then use a magnetic separator device to further refine the compound by removing any residual free annexin from the solution.
Using a protein assay. Quantify the A-N-X-S-P-I-O protein levels, then dilute to two milligrams per milliliter using 0.1 molar tris, HCL with 1%BSA and protease inhibitors and store at minus 80 degrees Celsius with PBS separately dilute the SPIO and A-N-X-S-P-I-O to an optical density of 0.1 and analyze using a dynamic light scattering or DLS machine. Ideally, the compound should produce mono modal peaks with a Z average particle size and poly dispersity index or PDI compare this size to the DLS measured size of the iron oxide nanoparticles alone, A-N-X-S-P-I-O.
Results that show a conjugate particle hydrodynamic diameter of 78 nanometers with a PDI of 0.13 reflect a homogenous species at a passage between 20 and 30 culture adult mouse bone marrow mesenchymal stem cells in DMEM supplemented with FBS and penicillin streptomycin next induce apoptosis in the cells by treating them with one millimolar docks for various times at 37 degrees Celsius. Label the cells with A-N-X-S-P-I-O or an equivalent weight of free SPIO for 15 minutes at 37 degrees Celsius. Wash the cells four times with PBS, an overlay with one milliliter of warm 1%aros to prevent diffusion of the SPIO particles.
Then proceed to MRI. Alternatively, after washing the cells, trypsin eyes them, place them in einor tubes and allow them to form pellets for MRI analysis. Use a 1.5 or three Tesla scanner with a standard knee receiver coil.
Place the culture dishes or tubes into an Agri phantom and use a gradient echo spoiled gradient sequence to image repetition. Time is set to 550 milliseconds. Echo time is 10 milliseconds.
Flip angle equals 15 degrees. Matrix is 2 56 by 2 56. Field of view equals three centimeters by three centimeters.
Slice thickness is 1.3 millimeters. Number of averages equals two and spacing is set to zero millimeters. Using regions of interest of a fixed area image axially through the cell layer on the plate for T two star signal loss adjust.
For background using a control ROI signal from the agri phantom. Finally, for each culture dish or tube, calculate the MRI contrast to noise ratio using the signal intensities or SI of the samples and control and standard deviation or SD of the background SI Measurement of T two star signal loss in vitro requires a homogeneous agri phantom and the lack of air bubbles, which will create signal void artifact. This artifact is difficult to distinguish from genuine T two star signal loss of SPIO and is critical to interpretable results shown here is an of an air artifact in pelleted neonatal cardiomyocytes as well as genuine iron oxide T two star signal loss.
It's important to apply an appropriate volume of agri gel to each culture dish so that sufficient axial MRI slices, which are approximately one millimeter thick can be made to adequately image the entire dish. This figure illustrates the expected MRI signal from individual culture plates in agar. There is typically some signal variation at the edges of the plate, so analysis of these uneven areas should be avoided.
After watching this video, you should have a good understanding of how to conjugate an EXIM protein with SPIO and evaluate its effectiveness as an in vivo and RI detectable apoptosis probe.
