Journal: Breastfeeding Medicine

Article Title: Phagocytosis of a Model Human Immunodeficiency Virus Target by Human Breast Milk Leukocytes Is Predominantly Granulocyte-Driven When Elicited by Specific Antibody

doi: 10.1089/bfm.2018.0232

Figure Lengend Snippet: ADCP activity by BM leukocytes requires actin dynamics and FcR engagement. The ADCP assay performed is based on the assay adapted from the study by Ackerman et al.49 ADCP target beads were incubated with dilutions of HIV V2i-specific human mAb 830A (black circles) or negative control anthrax-specific human mAb 3865 (gray squares). Plates were washed and 50,000 freshly isolated BM cells were added. For control experiments, actin inhibitor CytoD, FcBlock, or a combination of both was preincubated with cells before their addition to immune complexes. Cells were then treated, stained, and analyzed as in Figure 1. ADCP activity (bead-positive cells) was measured in the FITC channel. ADCP scores were calculated as follows: [(MFI of bead-positive cells) × (% of total CD45+ cells in the positive population)]. The data shown are from an experiment using BM sample 4 collected 31 days postpartum. ADCP, Ab-dependent cellular phagocytosis; CytoD, cytochalasin D; FcBlock, FcR-blocking agent; FcR, Fc receptor; HIV, human immunodeficiency virus; mAb, monoclonal antibody.

Article Snippet: For certain control experiments, 10 μg/mL of the actin inhibitor cytochalasin D (CytoD; Sigma), 50 μg/mL of FcR-blocking agent FcBlock (Becton Dickenson), or a combination of both was preincubated with cells before their addition to the plates.

Techniques: Activity Assay, Incubation, Negative Control, Isolation, Staining, Blocking Assay