The overall goal of this procedure is to generate macrophages from urine, bone marrow derived cell cultures. This is accomplished first by extracting the bone marrow from mouse hind legs. In the second step, the bone marrow resident macrophages are removed, and then the bone marrow cells are further cultured under macrophage differentiating conditions.
Then when the bone marrow cells have differentiated into macrophages, they can be collected for experimental analysis. Ultimately, confocal microscopy can be used to demonstrate the localization of bacterial LPS within bone marrow derived macrophage, end acidic compartments. The main advantage of this technique over existing methods like growing macrophages, cell lines, is that large number, so primary macrophages can be obtained.
Visual demonstration of method is critical as self production steps are difficult to learn and require practice, and to achieve success, Disinfect the skin of a euthanized mouse with 70%ethanol. Make an incision at the top of each hind leg and pull the skin down towards the foot to expose the muscle. Next, cut off the hind legs and use sterile scissors and forceps.
To remove the skin, place the legs in a sterile 35 millimeter Petri dish containing five milliliters of sterile ice. Cold PBS. Remove any skin and muscle attached to the bones, and then transfer the bones into a new sterile Petri dish containing five milliliters of ice.
Cold, sterile PBS. Wash the bones twice with five more milliliters of PBS and then transfer them into a sterile mortar containing five milliliters of ice. Cold, sterile PBS.
Now cut the tibia from the femur at the joint and use a pestle to gently smash the bones. Collect the supernatant in ice cold 15 milliliter tubes three times. Then filter the tissue suspension through a 70 micron nylon cell strainer.
To remove the solid fragments, spin down the filtrate and gently discard the supernatant. Dissociate the pellet with red blood cell lysis buffer, adding 20 milliliters of ice cold complete DMEM after 30 seconds to stop the reaction after centrifuging the cells, we suspend the pellet in 20 milliliters of 37 degrees Celsius, complete DMEM, and then split the cell suspension between two 100 millimeter Petri dishes. Incubate the dishes for four hours at 37 degrees Celsius, and then collect the supernatants into 50 milliliter tubes at room temperature.
After spinning down the supernatants, re suspend the pellet in 10 milliliters of complete DMEM supplemented with the previously prepared L 9 29 cell supernatant, and then filter the cells through a 40 micron nylon cell strainer. Add the filtrate to 140 milliliters of complete DMEM and L 9 29 cell media, and then distribute 10 milliliters of the cell suspension per dish into 15 100 millimeter Petri dishes. After incubating the cells for three days at 37 degrees Celsius and 5%carbon dioxide at 10 milliliters of complete DMEM plus L 9 29 supernatant and incubate the cells for an additional four days.
Monitoring the cell growth periodically with an inverted microscope to harvest the bone marrow derived macrophages first aspirate and discard the supernatant. Then wash the Petri dishes two times with complete DMEM. Next, add five milliliters of 37 degrees Celsius complete DMEM and use a rubber policeman to gently detach the cells, collect the bone marrow derived macrophages in 50 milliliter tubes, and then spin down the cells.
Resus suspending the pellets in 20 milliliters of complete DMEM. Finally, count the cells by triam blue exclusion. Using this method, large numbers of macrophages can be obtained in just a few days after isolating and plating the round non-adherent bone marrow cells minus the bone marrow resident macrophages as just demonstrated.
These representative BMDM precursor cells were incubated with G-M-C-S-F. After three days, the cells began to adhere and differentiate into macrophages. After seven days, a confluent monolayer of bone marrow derived macrophages was observed.
Flow cytometric analysis for F four 80 and H-L-A-D-R revealed that the bone marrow derived macrophages expressed both markers confirming the differentiation of the bone marrow cells into macrophages. To assess the PHA acidic capabilities of the bone marrow derived macrophages. The ability of the cells to internalize latex speeds was monitored as expected.
The number of beads per cell was determined to increase over time to evaluate their endocytic potential. The bone marrow derived macrophages were also co cultured with Coxiella. Burnett eye LPS in red was observed to be localized to a compartment that harbor's lysosomal associated membrane protein one in blue, but not cathepsin D in green, demonstrating that the LPS was present in the late endosomes, which are unable to fuse with lysosomes as expected bone marrow derived macrophage.
Bacterial pathogen interactions were further analyzed via their infection by TMA Wiley eye. As expected, the bacteria in red were not localized in the compartment that harbors KPS and D, which appears again in green. In this representative immuno blot, bone marrow derived macrophage signal transduction pathways were assessed demonstrating that bone marrow derived macrophages like tissue, isolated macrophages upregulate phosphorylated P 38 alpha map kinase levels in response to esia coli LPS stimulation.
This method can help answer key quests in the field of cellular microbiology, such as to which intracellular compartment micro localized.
