Scope of this study is to understand some of the limitations in the early life immune response to bacterial infection. In order to accomplish this, we need to develop and use research models that are specific to neonates. Isolation of bone marrow from a day 7 old pup is challenging and not well-established.
Neonatal bone marrow-derived macrophages are sensitive and prone to stress when compared to other BMDM. The technique used in this protocol is simple and reproducible. This method guides the users in isolating bone marrow from seven to nine day old neonatal mice without the use of any enzymes.
This study provides a standardized strategy to generate bone marrow-derived macrophages from neonatal mice that can be used to address questions about neonatal immunology. This reproducible method provides an age-specific model for progressing neonatal immune research across many areas of expertise. After decapitation, sterilize the body of the anesthetized neonate mouse with 70%ethanol.
Using forceps and fine tipped surgical scissors, make an incision between the abdomen and hind legs. Then remove the skin by pulling it toward the foot of the hind limbs. Scrape the connective tissue and muscle attached to the bones using sharp edged forceps.
Cut the tibia and femur with scissors. Use forceps to place the tibia and femur in a MEM culture media tube on ice. Transfer the bones to a 40 micron strainer with a collection tube.
Crush the bones with a three milliliter syringe plunger against the strainer to release the marrow. Centrifuge the cell suspension at 350g for five minutes at four degrees Celsius. Aspirate the supernatant and gently resuspend the cells in 0.2%sodium chloride to make a hypotonic solution for the lysis of erythrocytes.
Immediately add an equal volume of 1.6%sodium chloride to make the solution isotonic. Centrifuge the supernatant and remove the lysis solution. Resuspend the cells in complete DMEM and count the cells on an automated cell counter.
To begin, seed 20 million neonate mouse bone marrow cells per T75 flask in 10 milliliters of complete DMEM containing 2.5 milliliters of 10%L929-cell supernatant. Incubate the culture flask at 37 degrees Celsius with 5%carbon dioxide for five days. On day 3 of differentiation, add two milliliters of L929 conditioned media into the flask.
Under an inverted microscope with 20x magnification, daily observe the cells. To harvest the macrophages on day 5, aspirate the differentiation media from the flask. Wash the cells with five milliliters of PBS to remove non-adherent cells and serum proteins.
Add five milliliters of 0.05%Trypsin-EDTA to the flask and incubate at 37 degrees Celsius incubator with 5%carbon dioxide. After five minutes, add five milliliters of DMEM and pipette to detach the cells. Centrifuge the cell suspension at 350g for five minutes.
Dissociate the cell palate in one milliliter of complete DMEM. Count the cells on an automated cell counter. In the presence of L929-cell supernatant, bone marrow cells were differentiated into macrophages within five days.
Spindle-shaped cell formation was observed from the second day with nearly half displaying this morphology by day 3 and a majority by day 5. To begin, resuspend the mouse neonatal bone marrow-derived macrophages in complete DMEM without phenol red or antibiotics. Seed 500 microliters of cell suspension at a density of 200, 000 per quadrant in a 35 millimeter quad dish.
Remove the precalculated stock of Escherichia coli from 80 degrees Celsius. Aliquot the desired volume of cell suspension into a 1.7 milliliter micro centrifuge tube to prepare bacterial inoculum at a multiplicity of infection of 25. Then add PBS to a total volume of one milliliter.
Centrifuge the bacterial suspension at 2000g for five minutes and resuspend the pellet in 50 microliters of PBS. Add green fluorescent pH sensitive dye to a final concentration of 500 micromolars and incubate for 20 minutes in the dark for dye conjugation. Wash the bacteria four times with one milliliter of PBS by centrifugation at 1000g for five minutes.
Resuspend the pellet in 500 microliters of complete DMEM without phenol red and add the bone marrow-derived macrophage cultures. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide for four hours. Add 200 nanograms of cell permeable red fluorescent dye to stain the lysosomes.
Upon bacterial infection, abundant green fluorescent bacteria phagocytosis inside bone marrow-derived macrophages were detected. The green fluorescence further localizes with red fluorescence indicative of acidified lysosomes. The phagocytosis and appearance of green pH sensitive dye positive neonatal bone marrow-derived macrophages were also observed throughout the four hour infection.
