An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes
Transcription
For an ADCP assay, quickly invert the prepared ADCP plate to decant the supernatant after the final centrifugation, and add 5 x 104 freshly isolated breastmilk cells in 200 microliters of PBS plus BSA for a 2-hour incubation at 37 degrees Celsius.
At the end of the incubation, centrifuge the cells, and wash the pellets two times in 200 microliters of fresh PBS as demonstrated. After the last wash, stain the cells with 0.5 micrograms per milliliter of fixable viability stain, 450 per well, and 50 microliters of PBS for 30 minutes at room temperature, protected from light.
At the end of the incubation, pellet the cells for two washes in fresh PBS as demonstrated and stain the cells for the leukocyte markers of interest for 30 minutes at room temperature, protected from light. At the end of the incubation, collect the cells by centrifugation, and wash the cells two times in 200 microliters of 1% BSA plus PBS per wash.
After the last wash, fix the pellets in 200 microliters of 0.5% formaldehyde per well for 30 minutes at room temperature, protected from light. For flow cytometric analysis of the cell samples, set a flow cytometer equipped with a high throughput plate reader to withdraw 175 microliters of sample and to collect 5,000 cells per well.
Perform the initial gating to eliminate doublets and debris on a forward-scatter versus side-scatter plot. Use a side-scatter versus viability stain plot to eliminate the dead cells, and use a side-scatter versus CD45 plot to differentiate the major leukocyte classes.
For all CD45-positive cells, or for each leukocyte subset of interest, measure the antibody-dependent cellular phagocytosis activity by gating with a marker on the bead-positive cells in a histogram of the appropriate fluorophore channel for the conjugated beads. Then, calculate the antibody-dependent cellular phagocytosis scores as the median of the fluorescence intensity of the bead-positive cells by the percentage of the total CD45 plus the cells in the positive population.
