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 minus 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 2000 G for five minutes and resuspend the pellet in 50 microliters of PBS. Add green fluorescent pH sensitive dye to a final concentration of 500 micro molars and incubate for 20 minutes in the dark for dye conjugation.
Wash the bacteria four times with one milliliter of PBS by centrifugation at 1000 G 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 phagocytosed 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 dipositive neonatal bone marrow derived macrophages were also observed throughout the four hour infection.
