An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes

Published: April 30, 2024

Abstract

Source: Powell, R. L., et al. Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets. J. Vis. Exp. (2019).

This video demonstrates an assay to measure the antibody-dependent cellular phagocytosis (ADCP) by breast milk phagocytes. Fluorescently labeled microspheres conjugated to an antigen-antibody complex, are combined with a cell suspension isolated from breast milk to induce antibody-dependent cellular phagocytosis by the phagocytic leukocytes. Upon labeling the cells with antibodies against common leukocyte antigen (CD45), flow cytometry is used to measure the fluorescence intensity of microsphere-positive viable phagocytic leukocytes, indicating the level of ADCP activity.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Target Microsphere preparation Select a relevant target antigen. NOTE: In this example, the recombinant protein V1V2-2F5K was used, which was designed to mimic the trimeric apex of native HIV envelope. Perform biotinylation using a commercial kit (Table of Materials) according to the manufacturer…

Representative Results

Figure 1: Sample flow cytometry data of cells isolated from breast milk. Cells were processed and stained as described in the protocol. (A) Single cells were gated on to eliminate doublets in an FSC-H vs. FSC-A plot as shown, also gating out the small debris <5,000 in FSC-A. (B) This population was used to gate on live cells (which do not stain with the viabi…

Disclosures

The authors have nothing to disclose.

Materials

1 µm FluoSpheres NeutrAvidin-Labeled Microspheres Thermo Fisher F8776
BD Pharmingen PE Mouse Anti-Human CD45 BD 560975
Bovine serum Albumin MP Biomedicals 8810025
Corning V-bottom polystyrene 96-well plate Corning 3894
Cytochalasin D Sigma 22144-77-0
EZ-Link NHS-LC-LC-Biotin kit Thermo Fisher 21338
Falcon 15mL Conical Centrifuge Tubes Corning 352196
Falcon 50mL Conical Centrifuge Tubes Corning 352070
Fixable Viability Stain 450 BD 562247
Formaldehyde solution Sigma 252549
HBSS without Calcium, Magnesium or Phenol Red Life Technologies 14175-095
Human BD Fc Block BD 564219
Human Serum Albumin MP Biomedicals 2191349
Kimtech Science Kimwipes Delicate Task Wipers Kimberly-Clark Professional 34120
PBS 1x pH 7.4 Thermo Fisher 10010023
Polystyrene 10mL Serological Pipettes Corning 4488
Protein Concentrators PES, 3K MWCO, 0.5 mL Pierce 88512

Tags

Play Video

Cite This Article
An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes. J. Vis. Exp. (Pending Publication), e22130, doi: (2024).

View Video