How to: Bead-based Phagocytosis Assay

The bead-based phagocytosis assay offers a dynamic method to assess the process of phagocytosis — a fundamental aspect of the immune system’s defense mechanism. By utilizing fluorescently labeled beads as surrogate particles, this assay is a BSL-1/2-approved, flow cytometry-based method to study the mechanism of infection, especially Fc-receptor-mediated entry.

Reagents

• Suspension cells e.g. THP-1
• Latex beads, amine-modified polystyrene, fluorescent, 1 um. (Sigma, #L2778)
• MES hydrate
• Coating protein e.g. ovalbumin
• Antibodies for opsonization e.g. IgG
• Secondary, fluorophore-conjugated antibody
• Cytochalasin D
• Cell culture media e.g. RPMI 1640
• Human serum albumin (SAB) or fetal calf serum (FCS/ FBS)
• PBS
• 4% Paraformaldehyde (PFA)

All vendors are suggestions and are provided to look up manufacturer protocols.

Protocol

Preparation of antibody-opsonized Latex Beads:

1. Prepare the MES buffer: H20 with 25mM MES, pH6.
2. Wash the beads (2.5% solid, see manufacturers’ product sheet) twice with MES by resuspending and centrifugating at 3000 g for 20 minutes. Resuspend the beads to a final concentration of 1% solid in MES buffer.
3. Next, we coat our beads in protein. Therefore, add the beads to the protein dissolved in MES buffer (1 mg/ml) and incubate overnight at 4°C under constant rotation. This step ensures the correct orientation of the antibodies because the Fab-region will be bound to the protein, leaving the Fc-region open for interaction with Fc-receptors.
4. After centrifugation and another washing step, resuspend the pellet in MES buffer to 1% solid and add the beads to the antibody for opsonization. Again, incubate overnight at 4°C under constant rotation.
5. For final sample preparation, wash the beads in PBS and resuspend them to a final concentration of 10⁹ beads/ml. Store beads at 4°C.

Bead-based Phagocytosis Assay:

1. In a 96-well round-bottom plate, seed 1x10⁶ cells/ well in 2% FBS, or 5% SAB if using primary human cells, and antibiotic-free cell media (e.g. RPMI).
2. Optionally: Before adding the beads to the cells, pre-treat them for 30 minutes with 5 ug/ml Cytochalacin D at 37°C. Wash the cells with media afterward. This step served as an additional control because it inhibits actin polymerization and thus inhibits phagocytosis.
3. Add 10⁷ beads to the cells at a total volume of 100 ul per well and incubate for 1 h at 37°C.
4. Wash the cells with PBS and fixate them with 4% PFA for 15 minutes at room temperature (RT). This step essentially “freezes” the cells in place. After fixation, cells can also be stored at 4°C in FACS buffer (PBS with 3% FBS).
5. To differentiate between actually phagocytosed beads and those merely attached to the cell surface, stain the cells with a fluorophore-coupled secondary antibody. So if for example, your PE-fluorescent beads are opsonized with a rabbit IgG, stain with an anti-rabbit-APC antibody for 30 minutes at RT in FACS buffer.
6. After staining, wash cells twice with FACS buffer and resuspend in 200 ul FACS buffer.

Data Acquisition by Flow Cytometry:

1. First, gate on your cell population of interest (FSC-A vs. SSC-A).
2. On your cell population, gate for the single cells (FSC-A vs. FSC-H).
3. On your single cells, gate for the cells that have internalized or are attached to beads (PE vs. SSC-A). In some cases, you can even see multiple bead+ populations which result from cells taking up multiple beads.
4. On your bead+ population, gate for the cells that are negative for your secondary staining (PE vs. APC). These cells are those that have actually internalized the bead. This population should be less in the CytoD pretreated samples.
5. The final data can be represented in a “Phagocytic score” = (% bead+ cells x % internalized beads)*100.

Caution: All samples should be processed in an appropriate BSL cell culture hood.

Tips

• Which beads should I choose?
  Modifications: non-modified, amine-modified, sulfate-modified, carboxylate-modified….
  Size: diameter 0.1um — 2 um
  Color: non-fluorescent vs. fluorescent (red, blue, orange, yellow, green…)
  In general, check the literature on which beads have been used before for the kind of experiment you plan on doing. If you also plan to perform flow cytometry with these beads, maybe choose a fluorescent bead whose color fits with the rest of your stainings e.g. use a PE-fluorescent bead with an APC-coupled secondary antibody.
  In my experience, the manufacturers themselves are also very helpful and provide guidance on which bead to choose.
• How do I calculate the number of beads per ml (N)? S1 weight % solid of stock, pl density of spheres in suspension (g/ml), ps density of solid Sphere (g/cm3) = 1.055 g/cm3, d mean diameter (um) = 1 um.

• How do I calculate the amount of protein and antibody needed for surface saturation (S2)? C capacity of sphere for a given protein (mg protein/ m2 of sphere surface). C ovalbumin = 3 mg/m2; C IgG = 2.5 mg/m2.
  Equations and more information on bead preparation are provided by Bang Laboratories, Inc., especially in their TechNote 206. Many values (such as % solids, diameter, density, and surface area are provided on the certificate of analysis from the manufacturer.

• Which protein can I use to coat the beads? To coat the beads, I used ovalbumin, a cheap protein against which many different antibodies exist. Alternatively, if you have a specific protein against which you have, e.g. a human antibody, you use that too.
• Which antibody should I use to opsonize the beads? Ideally, we would want to use human antibodies when studying antibody-dependent cellular uptake into human cells. Sadly, human antibodies are limited, and expensive, and/ or the targeting protein is expensive/ not available. Thus, to study human Fcγ-receptor-mediated phagocytosis, I opsonized by beads in anti-ova IgG from rabbit or mouse.
• Always include negative controls in your phagocytosis experiments: a condition without beads and a condition with beads that are only covered in protein but not opsonized with antibodies.
• Add a condition not stained with the secondary antibody to set the gatting your secondary-negative population.
• Although I have no experience with adherent cells for this kind of assay, I am sure this protocol can be modified.