The overall goal of this procedure is to characterize Murine C1498 cells and the acute leukemia they induce after intravenous injection in mice. The main advantage of this procedure is that the murine myelomonocytic leukemia induced after a C1498 cells injection presents many features of the human disease. This animal model can help answer key questions in the Hematology field such as what are the mechanism that lead to leukemia development and immune deficiency.
The implication of this animal model extend toward the treatment of leukemia as it facilitates the testing of new chemo and immunothereputic agents. Begin by transferring a five to six week old female congenic C57 black 6 mouse into a sterile laminar flow hood. Then, place the animal in a restrainer.
Next, load a one millileter syringe equipped with a 29 gauge needle with 100 microliters of cells. Grasp the tail of the distal end and disinfect the tail skin with a 70%ethanol soaked gauze. Proceed to slowly inject one times 10 to the sixth cells into a lateral tail vein.
When all of the cells have been delivered remove the needle and apply gentle pressure at the sight of injection with sterile gauze. Then return the animal to its cage with careful monitoring for the next several weeks. 17 to 19 days later, insert a capillary tube into the medial canthus of the eye of the anesthetized leukemic animal.
100 to 200 microliters of blood will be drawn from the oribtal sinus into the capillary tube. Remove the tube and control the bleeding with the gentle application of a sterile gauze sponge onto the eye. Then, transfer the blood into an EDTA tube and store the tube on ice until the mononuclear cell isolation.
To isolate the blood mononuclear cells first add PBS supplemented with one millimolar EDTA up to a final volume of 500 microliters. Then, transfer the sample to a microcentrifuge tube. Next, use a one milliliter syringe equipped with a 30 gauge needle to carefully layer 500 microliters of separating solution under the blood, taking care not to mix the layers.
Separate the cells by centrifugation and use a pipette to transfer the opaque white mononuclear cell layer into a new microcentrifuge tube. Then, wash immune cells in one milliliter of PBS. Centrifuge them and re-suspend the pellet in 600 microliters of FACS buffer for staining, and analysis by flow cytometry.
To isolate the bone marrow place the mouse onto its back on a plastic board and secure the animals limbs. Disinfect the animal with 70%ethanol. Then, cut each leg at the top of the femur above the joint and gently pull on the tibias to disconnect them from the femurs.
Use forceps and scissors to remove the skin and muscles from the bones, and transfer the bones into a petri dish on ice. Use scissors to remove the extremities. Then insert a 26 gauge needle attached to a 10 milliliter syringe containing five milliliters of cold PBS into the end of one bone at a time and flush the bone marrows into the dish.
Pass the cell suspension through the needle tip several times to disrupt the bone marrow cells. Then, filter the cells through a new 70 micron filter into a 50 milliliter conical tube to obtain a single cell suspension. Centrifuge the cells and re-suspend the pellet in two milliliters of lysis buffer.
Immediately fill the tube with up to 50 milliliters of cold PBS and centrifuge the cells. Then, re-suspend the bone marrow pellet at one times 10 to the sixth cells per milliliter in cold FACS buffer. To prepare the bone marrow cells for staining first, place slides into disposable chambers with pre-attached filter cards and place the chambers into a cytocentrifuge.
Add 100 microliters of FACS buffer to each filter card chamber and briefly centrifuge the slides. At the end of the spin add 100 microliters of cells to each chamber and centrifuge the cells. Then, carefully remove the slides from the chambers for air drying.
To stain the bone marrow cells immerse the slides in a coplin jar containing May-Grunwald solution for five minutes. Next, transfer the slides into a jar of buffer solution with a pH of 6.8 for one minute. Then, transfer the slides into Giemsa R solution for 10 minutes.
Followed by a 10 second wash with pH neutral water. After the wash, allow the slides to air dry and apply one drop of mounting medium to each sample. Then, place one edge of a cover glass onto the slide and carefully lower the cover glass onto the cells pressing gently to remove any air bubbles.
Compared with control animals the C1498 cell injected animals display a massive infiltration of C1498 cells in their bone marrow as observed by the blast-like appearance of the cells. The bone marrow infiltrating C1498 cells demonstrate monocytic and granulocytic phenotypes indicative of monoblastic and myeloblastic cells respectively, characteristic of acute myelomonocytic leukemia. Flow cytometric analysis of the mononuclear cell frequencies in blood samples from the C1498 injected mice indicates that approximately a quarter of the whole white blood cell population is made up of leukemic cells.
T and B lymphocyte frequencies are similar between control and C1498 injected animals. Monocyte frequencies, however, are observed at higher levels in C1498 injected mice, a feature of acute myelomonocytic leukemia. When performing the cytochemistry and May-Grunwald Giemsa procedures it is important to remember to maintain the appropriate pH values, temperatures, and incubation times.
After watching this video you should have a good understanding of how to characterize acute myelomonocytic leukemia. After its development, this technique paved the way for researchers in the field of Hematology to explore the nature of spontaneous and induced acute leukemia in other animal models.
