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

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

1. 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.
2. Perform biotinylation using a commercial kit (Table of Materials) according to the manufacturer's protocol.
   1. Calculate mmol of the biotin reagent to add to the reaction for a 5-fold molar excess using the formula: mmol biotin = mL protein....

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 gatin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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