Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

We developed a rigorous method for isolating cells from human breast milk to study antibody dependent cellular phagocytosis, or ADCP, by breast milk phagocytes against HIV targets. This technique facilitates the relatively quick and easy isolation of breast milk cells to evaluate the capacity of leukocyte populations to perform ADCP. Demonstrating the procedure will be Alisa Fox, a technician from my laboratory.

After selecting a relevant target antigen, use a commercial kit to biotinylate the protein of interest according to the manufacturer's protocol. And deposit the biotinylated sample into the upper chamber of a spin column. Add PBS up to 400 microliters and cap the chamber before inserting the column into a collection tube for centrifugation.

After discarding the flow through, add PBS to 400 microliters for a second centrifugation. Discard the flow through, add PBS to 400 microliters, and measure the protein concentration by a spectrophotometer to conjugate the biotinylated protein to micrometer microspheres. Incubate six micrograms of protein with 12 microliters of stock beads in 200 microliters of PBS per plate of conjugated beads at room temperature for two hours, with gentle vortexing every 20 minutes.

At the end of the incubation, pellet the protein conjugated beads by centrifugation and discard the supernatant. After gentle vortexing, resuspend the protein in 1200 microliters of 0.1%BSA in PBS, and wash the tube by centrifugation two more times as just demonstrated. After the last wash, resuspend the pellet in 1200 microliters in PBS plus BSA.

To set up ADCP Assay Plates, first prepare 12 microliter dilutions of antibody, or the immune serum of interest, in 2%BSA in PBS in a round bottom 96 well plate. Add 10 microliters of protein conjugated beads per well to the plate for a two hour incubation at 37 degrees Celsius. At the end of the incubation, add 200 microliters of PBS plus BSA.

After obtaining human breast milk from healthy lactating women expressing using a pump, within four hours of expression separate the milk contents by centrifugation and carefully pour off the skim milk and fat, leaving the cell pellet undisturbed. Wipe the inside of the tube with a lint-free wipe to remove all of the fat from the tube wall and add 10 milliliters of PBS plus HSA. Resuspend the pellet with gentle pipetting to avoid cell activation in the apoptosis, and transfer the cell suspension to a 15 milliliter tube for centrifugation.

After a second wash in PBS plus HSA as just demonstrated, gently resuspend the cells in one to two milliliters of fresh PBS plus HSA for counting. For an ADCP Assay, quickly invert the prepared ADCP plate to decant the supernatant after the final centrifugation and add five times 10 at a fourth freshly isolated breast milk cells in 200 microliters of PBS plus BSA for a two 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 in 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 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 5000 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. Here, the gating strategy for eliminating doublets, debris, and dead cells is shown. Between one to 12%of total live cells are typically CD45 positive leukocytes with most of the purported side scatter low to intermediate CD45 low monocytes appearing to be precursors, or to immature cells, based on the side scatter versus CD45 gating.

The purported granulocytes are defined as side scatter high, CD45 intermediate cells. Measurement of the ADCP activity of freshly isolated breast milk cells using an HIV specific human monoclonal antibody at one month postpartum reveals that treatment with actin inhibitor Cytochalasin D and or Fc receptor blocking antibodies prior to incubation with antibody bound antigen coupled beads results in the ADCP activity at or below that observed in the presence of control antibody. The ADCP score determined for total CD45 positive cells is commonly between 25 to 35 fold above the background levels, defined using a negative control, anti-anthrax monoclonal antibody.

Every breast milk sample is unique and some pellets may be hard to see. In addition, the leukocyte populations, and therefore the ADCP scores, may vary immensely from sample to sample.