Nov 05, 2025
Opsonic Phagocytosis Assay using the THP-1 Monocytic Cell line and Antigen-Coated Beads
- Kaitlin Pekin1
- 1Burnet Institute
Protocol Citation: Kaitlin Pekin 2025. Opsonic Phagocytosis Assay using the THP-1 Monocytic Cell line and Antigen-Coated Beads. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19pwylr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 07, 2024
Last Modified: November 05, 2025
Protocol Integer ID: 109321
Keywords: opsonic phagocytosis, THP-1 cells, antibody dependent cellular phagocytosis, THP-1 monocytes, phagocytosis of antigen, opsonic phagocytosis assay, phagocytic cell, phagocytosi, level of phagocytosi, antibody, flow cytometry the fluorescent intensity, antigens of interest, opsonised antigen, antigen, immune complex, ability of antibody, fcγ receptor, using flow cytometry, monocytic cell line, neutrophil, modified fluorescent latex bead, fluorescent latex bead, coated bead, polyclonal serum as source, polyclonal serum, rabbit serum sample, beads this protocol, coupled bead
Abstract
This protocol is designed to quantify the ability of antibodies to mediate phagocytosis of antigen-coated beads. We leverage the well-established THP-1 monocytic cell line as a model for phagocytic cells and use polyclonal serum as source of antibodies. Briefly, (i) antigens of interest are coupled onto amine-modified fluorescent latex beads, (ii) the antigen-coupled beads are then opsonised with antibodies (e.g. human or rabbit serum samples), (iii) the opsonised antigen-coupled beads are incubated with THP-1 cells to allow for phagocytosis, (iv) using flow cytometry the fluorescent intensity and proportion of THP-1 cells that have taken up opsonised beads is measured to quantify the level of phagocytosis.
Note: The THP-1 monocytic cell line highly expresses Fcγ receptor I (FcγRI), that binds monomeric IgG and not FcγRIIa and FcγRIII expressed by neutrophils that bind to immune complexes.
Materials
Consumables:
50 mL and 10 mL Falcon tubes
Reagent reservoirs
Eppendorf Tubes (1.5 mL)
U-bottom plates (Corning Cat# 353077)
Glass test tubes
FACS tubes
Equipment:
Centrifuge with plate insert buckets
Flow Cytometer (Canto or Similar, with HTS and plate compatibility)
Incubator (5% CO2 at 37 degrees Celsius)
Cell Counter (Countess) or Hemocytometer
96-well plate washer (optional)
Sonicator
Vortex
Glass microscope slides and coverslip
Microscope 100x lens
Reagents:
Amine Bead Coupling:
Amine-modified fluorescent latex beads (2.0 μm, Sigma-Aldrich, Cat# L9529-1mL)
Wash Buffer- 0.01M PBS and 0.1% SDS
Glutaraldehyde Solution- 8% Glutaraldehyde in Wash Buffer
Protein Solution- Protein at 0.5 mg/mL diluted in Wash Buffer
Quenching Solution- 0.15 mL ethanolamine in 4.8 mL Wash Buffer
Blocking Solution- 10 mg/mL BSA, 0.01M PBS and 0.1% SDS
Storage Solution- 5% Glycerol, 0.1% Sodium Azide in Blocking Solution
1mM EDTA in 1x PBS
Phagocytosis Assay:
1% BSA in 1xPBS - Filter sterile
1x PBS 0.05% Tween
Antigen Coupled Beads - Amine beads coated with recombinant antigen/peptide at 5 x 107 beads/mL
THP-1 Cells - Diluted to 5 x 105 cells/mL, Obtained from commercial cell line database
THP-1 Cell Incomplete Media - RPMI (without glutamine, 500 mL) + 5 mL Penicillin-Streptomycin-Glutamine
THP-1 Cell Complete Media - THP-1 cell Incomplete Media + 10% Foetal Calf Serum (heat inactivated), sterile
Serum - Neat or diluted serum, heat inactivated
Fixative- 2% Paraformaldehyde diluted in 1% BSA in 1x PBS
Trypan Blue Dye
Troubleshooting
Safety warnings
Steps involving serum and cells should be completed in a biosafety cabinet. Steps involving paraformaldehyde should be completed in a fume hood. Consideration into waste disposal and decontamination is also required based on relevant guidelines.
Covalent Coupling of Antigen to Fluorescent Beads
6h 35m
Add 100 μL of Amine-modified fluorescent latex beads to 400 μL of Wash Buffer in an Eppendorf tube
Note
Ensure beads are well resuspended before transfer, vortex or sonicate beads for approximately 30 seconds.
Beads are light sensitive should be covered from light at all times e.g. dark tubes, covering in foil or storing in a black container.
Centrifuge beads at1500 x g, 00:05:00
5m
Remove supernatant and resuspend in 600 μL Wash Buffer
Centrifuge beads at1500 x g, 00:05:00
Remove supernatant and resuspend beads in 400 μL of Glutaraldehyde Solution
Incubate on a vortex, with tube attachment Overnight at Room temperature
Wash beads (procedure described in steps 4.1-4.3)
Centrifuge beads at1500 x g, 00:05:00
Remove supernatant and resuspend in 600 μL Wash Buffer
Centrifuge beads at1500 x g, 00:05:00 and remove supernatant
5m
Resuspend in 100 μL of Protein Solution and incubate on a vortex 05:00:00 at Room temperature
Note
Beads can also be coupled with Streptavidin to allow for the use of biotinylated proteins/peptides
5h
Centrifuge beads at1500 x g, 00:05:00 and remove the supernatant. Resuspend with 600 μL of Quenching Solution
5m
Incubate on a vortex 00:30:00 at Room temperature
30m
Centrifuge beads at1500 x g, 00:05:00 and remove the supernatant. Resuspend with 600 μL of Blocking Solution
Incubate on a vortex Overnight at Room temperature
Centrifuge beads at1500 x g, 00:05:00 and remove the supernatant. Resuspend with 1200 μL of Storage Solution
Transfer beads to glass tubes and sonicate for 00:30:00 in an ice slurry
30m
Visualise separation of beads by microscopy, add 2 μL of beads to a microscope slide and add a coverslip, using 100x magnification
Note
If beads are not well separated (clumps of beads together) and there is a low percentage of single beads repeat the sonication step
Determine concentration of beads using hemocytometer or flow cytometry with counting beads
Procedure for counting beads with hemocytometer:
Vortex beads for 30 seconds to ensure proper resuspension. Into an Eppendorf tube add 3 μL of coupled beads to 600 μL of 1 mM EDTA in 1x PBS (1:200 dilution).
Vortex tube and transfer 10 μL under the coverslip of a hemocytometer
Using 100x lens, focus the slide and leave the beads to settle for 00:15:00 before counting
15m
Count the outer 4 squares, excluding beads on the outer boundaries (see image below)
Determine the average of the 4 squares
Multiply the average x dilution factor x 104
Image obtained from StemCell Technologies, "How to Count Cells with a Hemocytometer"
Procedure for counting with flow cytometry:
Combine 2 μL of coupled beads with 20 μL of counting beads in 600 μL of 1 mM EDTA in 1x PBS into a FACS tube
Completed required start-up procedures on the flow cytometer.
Set up 2 Histograms
Population #1: Histogram displaying count and the APC channel (Counting Beads, Log Scale), capped to 2,000 events
Population #2: Histogram displaying count the PE-A channel (Coupled Beads, Log Scale)
Acquire samples and set a stopping gate of 2,000 events on the Population#1
Determine the concentration of beads by:
Value from Population #2 / 2,000 (from Population #1) x (dilution factor) x 106
Dilute beads in Storage Solution to a concentration of 5 x 107 beads/mL and store away from light at 4 °C
To protect from light exposure- wrap the Falcon tubes in foil, or use dark Eppendorf tubes and store in a black container
Determine Concentration of THP-1 Cells
Using a Pasture Pipette resuspend flask of THP-1 cells (monocytes) and transfer a small sample into an Eppendorf tube
Add 10 μL of resuspended cells to 10 μL of Trypan Blue into another eppendorf tube and mix well
Transfer 10 μL of solution to preferred cell counting method:
Cell Counter (e.g. Countess) or Hemocytometer
Cell Counter: Load samples into compatible chamber slides and count following manufacturer's instructions
Hemocytometer: Same as described previously, cells do not require the 'settling' step.
Determine the concentration and viability of THP-1 cells in the sample
Expected result
Cell concentration should be at least 5 x 105 cells/mL in order to complete the assay, a concentration around 7 x 105 cells/mL or higher is ideal
Cell viability should be above 95%
Prepare THP-1 Cells
After determining the concentration of THP-1 cells, determine the required volume of cells needed for the assay and number of samples to be run
Volume of cells required = 150 μL/well * number of wells
Concentration of cells required = 5 x 105 cells/mL
= (5 x 105 cells/mL * volume of cells required) / actual concentration of cells
Dilute cells to the required volume in a Falcon tube using THP-1 Complete Media
Store diluted cells in an incubator at 37 °C with 5% CO2 until required for the assay
Phagocytosis Assay
3h 1m 30s
Block required number of U-bottom plates with 300 μL/well of 1% BSA in 1x PBS 00:30:00 at Room temperature
30m
Wash plates thrice using a plate washer or manually using 1x PBS 0.05% Tween
Tap out excess and ensure plates are completely dry
Prepare required sera for assay and dilute in 1% BSA in 1x PBS (total volume required 50 μL per sample)
Note
The dilution of serum samples for this step will need to be optimised to determine the best signal:background level.
Ensure "no serum" blank controls are included in the plate layout
Assay can be completed using a range of sera types: human sera, monoclonal antibodies, bleeds from vaccinated mice or rabbits
Vortex pre-prepared antigen-coated beads for 00:00:30
30s
Add 20 μL/well (or the volume needed to obtain the optimal ratio beads:cells) of desired antigen-coated beads (at a concentration of 5 x 107 beads/mL)
Note
The ratio of beads:cells may need to be optimised, recommended ratio is 6.66 beads:cell
Wash the plate thrice by centrifugation, following procedure in steps 31.1-31.4
Centrifuge plate at 500 x g for 00:02:00 at Room temperature using centrifuge plate inserts
2m
Quickly flick plate to remove supernatant
Note
A quick inversion of the plate to remove supernatant is required to ensure the beads do not get lost, avoid a slow/gentle pour of the plate.
Resuspend beads with 20 μL/well of 1% BSA in 1x PBS and mix by pipetting up and down 10x
Repeat 2x to total three washes
After the final wash resuspend beads in 20 μL/well of 1% BSA in 1x PBS
Add 50 μL/well of pre-diluted serum to the U-bottom plate containing washed beads and mix by pipetting up and down 10x
Note
If using biotinylated proteins/peptides with streptavidin coated beads, incubate beads with peptide for an hour at room temperature prior to adding serum. The amount of protein/peptide to add will also need to be optimised
Incubate plate at Room temperature for 01:00:00 covered in foil
1h
Wash the plate thrice by centrifugation, following procedure in steps 34.1-34.4
Centrifuge plate at 500 x g for 00:02:00 at Room temperature Using centrifuge plate inserts
Quickly flick plate to remove supernatant
Resuspend beads in 150 μL/well of 1% BSA in 1x PBS and mix by pipetting up and down 10x
Repeat 2x for a total of three washes
After the final wash resuspend beads in 100 μL/well of THP-1 Cell Complete Media and mix by pipetting up and down 10x
Mix wells again by pipetting up and down 10x and split each well into duplicate wells by transferring 50 μL into separate wells/rows of a new U-bottom blocked plate
Add 150 μL/well of THP-1 cells at 5 x 105 cells/mL diluted in THP-1 Cell Complete Media and mix gently by pipetting up and down 10x avoiding air bubbles
Incubate plate for 00:40:00 wrapped in foil, at 37 °C with 5% CO2
40m
Pre-cool centrifuge to 4 °C
Stop phagocytosis by centrifuging plate 300 x g for 00:04:00 at 4 °C
Alternatively, place plate in a 4 °C fridge for 00:30:00 then centrifuge at 300 x g for 00:04:00
34m
Quickly flick plate to remove supernatant
Resuspend cells in 50 μL / well of 2% fixative and mix by pipetting up and down 10x
Incubate at 4 °C in the dark for 00:15:00
15m
Add 100 μL/well of 1% BSA in 1xPBS then wash twice using centrifugation, following procedure in steps 40.1-40.4
Centrifuge plate at 500 x g for 00:02:00 at Room temperature using centrifuge plate inserts
Quickly flick out supernatant
Resuspend cells in 150 μL/well of 1% BSA in 1x PBS and mix by pipetting up and down 10x
Repeat 1x for a total of two washes
After the final wash resuspend cells in 100 μL/well of 1% BSA in 1x PBS and mix by pipetting up and down 10x
Quantify the level of phagocytosis by flow cytometry (e.g. FACS CantoII, BD Biosciences), ensure there are no air bubbles in the individual wells before reading the plate
FACS Acquisition
Completed required start-up procedures on the flow cytometer.
Set up the gating procedure to calculate the percentage of THP-1 cells that have phagocytosed the fluorescent beads
Note
The required FACS machinery used will need to be compatible with plates (HTS) and require relevant lasers
Gate#1 FSC-SSC Plot
Gate the entire population of THP-1 cells as gate #P1
Both FSC and SSC Scales set as Log Scale
Gate#2 Select Single Events
Add an additional FSC and SSC plot and apply gate #P1 and gate single events only as gate #P2 (removed doublets)
Both FSC and SSC scales set as Log Scale
Gate#3 Gate Phagocytosed Cells
Add a histogram displaying Count and PE-A (Log Scale), gate all phagocytosed events as gate #P3
The negative samples will appear closer to 0. The ideal range for phagocytosed cells is between 103 and 105
When acquiring the sample, set the stopping gate to #P1 and number of events to record as 10,000
Note
The ideal voltages will need to be optimised with a positive control to exclude background fluorescence
Process FACS files using Flowjo Analysis software to obtain the percentage phagocytosis index (%PI)
Percentage phagocytosis calculated by dividing the number of THP-1 cells with phagocytosed beads by the total population of THP-1 cells.
Protocol references
Feng et al. 2021. Mechanisms and targets of Fcgamma-receptor mediated immunity to malaria sporozoites. Nat Commun.
Osier et al. 2014. Opsonic phagocytosis of Plasmodium falciparum merozoites: mechanism in human immunity and a correlate of protection against malaria. BMC Med.
Feng et al. 2022. Induction, decay, and determinants of functional antibodies following vaccination with the RTS,S malaria vaccine in young children. BMC Med.
Kurtovic et al. 2021. Multifunctional Antibodies Are Induced by the RTS,S Malaria Vaccine and Associated With Protection in a Phase 1/2a Trial. J Infect Dis.
Acknowledgements
We acknowledge Gaoqian Feng for developing the original protocol and training. Monash University ARAFlowcore for assistance using the BD analysers and protocol development. Amaya Ortega pajares for assistance and initial advice. NHMRC for project funding.