Determination of BV2 Microglial Phagocytic Activity (Fluorescent Microspheres / FluoSpheres Assay; Flow Cytometry/Microscopy)

Determination of BV2 Microglial Phagocytic Activity (Fluorescent Microspheres / FluoSpheres Assay; Flow Cytometry/Microscopy)

Overview

This assay quantifies phagocytosis of fluorescent carboxylate-modified microspheres by BV2 microglia. After exposure to the test compound, cells are incubated with FluoSpheres, washed to remove non-internalized beads, detached with Accutase containing Hoechst 33342 to identify nuclei, and analyzed by flow cytometry. Phagocytosis is reported as the percentage of FluoSphere-positive cells normalized to the control.

Materials and reagents

• BV2 microglial cells
• Culture medium appropriate for BV2 cells 
• FluoSpheres™ Carboxylate-Modified Microspheres, 2.0 µm, red fluorescent (580/605), 2% solids (Thermo Fisher Scientific, F8826)
• Cytochalasin D (Sigma-Aldrich, C8273-1MG)
  • Stock: 1 mM in DMSO
  • Working concentration: 2 µM
• DMSO, anhydrous 99.9% (Sigma-Aldrich, 276855)
• Hoechst 33342 (Invitrogen, H1399), 2 µg/mL in Accutase solution
• Accutase solution (Sigma-Aldrich, A6964)
• PBS (room temperature)
• 12-well tissue culture plate
• Vortex, sonicator (bath or probe), microcentrifuge capable of 10,000 × g
• Flow cytometer/confocal microscope

Controls (recommended)

1. Vehicle control (e.g., DMSO at the same final concentration as in treated groups)
2. Cytochalasin D control (phagocytosis inhibition): 2 µM, added 30 min before FluoSpheres
3. 4 °C incubation control (binding/uptake control): FluoSpheres incubation performed at 4 °C for 2 h

Procedure

A. Cell seeding

1. Seed BV2 cells in a 12-well plate at 3.2 × 10⁴ cells/cm² in 1.0 mL of complete culture medium per well.
2. Incubate for 24 h under standard conditions (37 °C, 5% CO₂).

B. Treatment with test compound

1. Add the test compound and continue incubation under standard conditions.
   • If medium exchange is performed, state it consistently (recommended: either always change medium or never change medium, unless the experimental design requires otherwise).
   • Include vehicle control matched for medium change and solvent concentration.

C. FluoSphere incubation (phagocytosis step)

1. Prepare FluoSpheres working suspension (Section “FluoSphere preparation”) immediately before use and protect from light.
2. Add FluoSpheres to each well at 50 µL per 1.0 mL of culture medium.
3. Incubate for 2 h at 37 °C (standard condition).
   • Cytochalasin D control: add 2 µM cytochalasin D 30 min before FluoSpheres.
   • 4 °C control: incubate cells with FluoSpheres for 2 h at 4 °C (keep all solutions cold during this step).

D. Washing (remove non-internalized beads)

1. Wash cells 3× with 1.0 mL PBS per well to remove non-phagocytosed / non-internalized microspheres.
   • Aspirate carefully to avoid detaching cells.

E. Detachment and nuclear staining

1. Add Accutase containing Hoechst 33342 (final 2 µg/mL) to each well (volume sufficient to cover the well bottom; typically 300–500 µL for a 12-well).
2. Incubate for 15 min at 37 °C, protected from light.
3. Gently resuspend to obtain a single-cell suspension and transfer cells to flow cytometry tubes.

Flow cytometry acquisition and analysis (BD FACSCanto II; FACSDiva v6)

Suggested gating strategy

within Hoechst-positive cells, determine the % of FluoSphere-positive cells (red fluorescence channel appropriate for 580/605 beads; use the instrument’s configuration/filters to select the correct detector).

Calculation

Phagocytosis index

index = % of FluoSpheres-labelled cells in analysed group/% of FluoSpheres-labelled cells in the control.

Control group = vehicle-treated (and matched medium change condition).

FluoSphere preparation (volumes for 2 wells; prepare fresh; protect from light)

1. Mix FluoSpheres thoroughly by gentle inversion (do not vortex the stock vial aggressively unless recommended by the manufacturer).
2. Transfer 2 µL of FluoSpheres stock into 1.0 mL of sterile ultrapure H₂O.
3. Vortex 30 s.
4. Sonicate to disrupt aggregates (e.g., 2 min gentle sonication). Confirm dispersion under a microscope if possible.
5. Centrifuge at 10,000 × g for 15 min.
6. Carefully remove supernatant.
7. Resuspend the pellet in 100 µL of culture medium.
8. Vortex 30 s and sonicate again under the same conditions.
9. Pool the prepared suspensions to obtain a single, homogeneous FluoSpheres working suspension for multiple wells, if required.
10. Add 50 µL of this suspension to 1.0 mL medium per well.

Reagent preparation

• Cytochalasin D: 1 mM stock in DMSO; dilute into culture medium to 2 µM final.
• Hoechst 33342: add to Accutase solution to 2 µg/mL final.

References

• Matuszewska M, Cieślik M, Wilkaniec A, Strawski M, Czapski GA. The Role of Bromodomain and Extraterminal (BET) Proteins in Controlling the Phagocytic Activity of Microglia In Vitro: Relevance to Alzheimer's Disease. 
  Int J Mol Sci. 2022 Dec 20;24(1):13. doi: 10.3390/ijms24010013.
• Matuszewska M, Wilkaniec A, Cieślik M, Strawski M, Czapski GA. The Inhibition of Bromodomain and Extraterminal Domain (BET) Proteins Protects Against Microglia-Mediated Neuronal Loss In Vitro. Biomolecules. 2025 Apr 4;15(4):528. doi: 10.3390/biom15040528.