A Lactate Dehydrogenase Release Assay to Detect Cryptococcus neoformans Infection in Macrophages
Transcript
To prepare fungal cells for a macrophage infection, collect and centrifuge C. neoformans cells from a mid-log phase culture, followed by three gentle washes with 1 milliliter of room temperature PBS under the same centrifuge conditions.
After the last wash, re-suspend the fungal cells at a 1.2 times 10 to the eighth cells per milliliter, of antibiotic-free cell culture medium concentration, and add 1 milliliter of fungal cell suspension to each of 4 wells of a 70% to 80% confluent 6-well macrophage culture plate. Then, place the plate in the cell culture incubator for three hours. Carefully tilt the plate to allow each well to be gently washed three times with 1 milliliter of PBS per well per wash.
To measure the infection proficiency after the last wash, add 1 milliliter of antibiotic-free medium to each well of the 6-well plate, and place the plate in the cell culture incubator. At the appropriate experimental time points, collect the supernatant from each well, and measure the amount of LDH in each well according to standard protocols. At the same time points, lyse the uninfected macrophage cells to determine the value for maximum cytotoxicity.
The cytotoxicity can then be calculated using the formula as indicated.
