The overall objective of this procedure is to follow in real time the formation of proplatelet from mouse megakaryocytes, which matured in their native environment. This method has the advantage of being simple, reproducible, and fast. One can analyze many megakaryocytes and visualize the formation of proplatelets in only six hours, instead of waiting for the first day of culture as for mouse megakaryocytes in vitro.
An interesting application of this method is a possibility to study the effect of the pharmaceutical treatment or genetic mutation, specifically in the step of proplatelet extension. Here there are no interference with the differentiation process, as in the case of in-vitro culture. This video helps ensure these key steps of flushing and cutting the bone marrow.
It's also explained how to classify the morphology and megakaryocytes, which is really useful for the characterization of defects in thrombophilic disease. To begin, warm Tyrode's buffer at 37 degrees Celsius, and turn on the heating chamber of the microscope to bring the temperature to 37 degrees Celsius. Prepare all necessary tools, such as a timer, incubation chambers, five milliliter 21 gauge syringes, forceps, a razor blade, Pasteur pipettes, glass slides, and 15 milliliter centrifuge tube.
Flush the bone marrow with two milliliters of Tyrode's buffer by introducing a 21 gauge needle into the opening of the femur on the knee side, and slowly press the plunger to retrieve an intact marrow cylinder. Use a three-minute liter plastic pipette to carefully and gently transfer the intact bone marrow onto a glass slide. Cut off the ends of the marrow that may have been compressed at the time of the flush.
Then cut thin transversal sections of 0.5 millimeter thickness using a sharp razor blade to avoid damaging the megakaryocytes. Using a plastic pipette, collect 10 sections into a one milliliter tube containing Tyrode's buffer. Carefully transfer the sections to an incubation chamber with a diameter of 13 millimeters.
Aspirate the buffer and adjust the volume to 30 microliters of Tyrode's buffer supplemented with 5%mouse serum. Position the sections at a distance. Seal the self adhesive chamber with a 22 by 55 millimeter cover slip, inclining the cover slip to avoid the formation of air bubbles.
Place the chamber in the heating chamber at 37 degrees Celsius. Start the chronometer and run the experiment for six hours at 37 degrees Celsius, Make videos to record the transformation of the megakaryocytes. After 30 minutes, the marrow cells gradually migrate to the periphery of the explant forming a mono layer.
After one hour of incubation, megakaryocytes can be identified by their large size and polylobulated nuclei. The number of megakaryocytes increases after three hours of incubation, and some have long extensions. Draw a map to localize each section in the incubation chamber.
After one hour, identify the visible megakaryocytes, which are giant polylobulated cells on each section's periphery, and plot their positions on the drawing. Repeat this procedure after three and six hours. Megakaryocytes are counted manually and classified according to their morphology at three and six hours after sealing of the incubation chamber.
They are classified as small, large with thick intention, and proplatelet extending. With the help of mapping, their evolution can be followed over time. The results are expressed as a percentage of each class at each observation time.
Classically, half of the megakaryocytes visible at the periphery extend proplatelets at six hours for wild type mouse bone marrow. The fate of round megakaryocytes was followed by capturing sequential images over time to image how they form proplatelets. An important thing to remember when attempting this procedure is to keep the explants at 37 degrees Celsius for the duration of the experiment.
A different temperature can change the results. Interestingly, the explant protocol can be used after introverted microscopy experiments. In the end the megakaryocytes in the bone marrow explants will be naturally fluorescent, and their behavior can be easily investigated.
This make it possible to combine different treatment on the same animals and thus reduce the number of mice. In conclusion, the explant method is fast, simple, and provide many information on the capacity of natural megakaryocytes to external proplatelets. The resulting qualitative and quantitative finding are key to our understanding of thrombophilic disease.
in a physiological or pathological context.
