Protocol for assessing anti-Siglec-9 antibody-mediated tumor phagocytosis by macrophages using pH-sensitive dyes and flow cytometry

Summary

Here, we present a protocol to assess and quantify macrophage-mediated cancer cell phagocytosis using pH-sensitive dyes and flow cytometry. We describe steps for isolating and sorting CD14⁺ monocytes from leukocyte reduction system (LRS) cones, differentiating monocyte-derived macrophages, and preparing K562 leukemic cells for co-culture. We then detail procedures for quantifying phagocytic activity using flow cytometry and pH-sensitive dyes and visualizing internalized K562 cells using a fluorescence microscope. This technique enables the study of macrophage phagocytic function and supports application in cancer immunotherapy research.

For complete details on the use and execution of this protocol, please refer to Muneekaew et al.¹

Graphical abstract

Highlights

• •Steps for isolating and sorting CD14⁺ monocytes from leukocyte reduction system cones
• •Instructions for differentiating macrophages and preparing K562 cells for co-culture
• •Guidance on quantifying phagocytic activity using flow cytometry and pH-sensitive dyes
• •Visualization of internalized K562 cells within macrophages using fluorescence microscope

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol to assess and quantify macrophage-mediated cancer cell phagocytosis using pH-sensitive dyes and flow cytometry. We describe steps for isolating and sorting CD14+ monocytes from leukocyte reduction system (LRS) cones, differentiating monocyte-derived macrophages, and preparing K562 leukemic cells for co-culture. We then detail procedures for quantifying phagocytic activity using flow cytometry and pH-sensitive dyes and visualizing internalized K562 cells using a fluorescence microscope. This technique enables the study of macrophage phagocytic function and supports application in cancer immunotherapy research.

Before you begin

The following section outlines a step-by-step protocol for evaluating the phagocytic activity of monocyte-derived macrophages (MDMs) against K562 leukemic cells. This protocol takes approximately 10 days to complete phagocytic assay. The workflow comprises four steps: (1) Isolation of CD14⁺ monocytes from a leukocyte reduction system (LRS) cone, (2) Differentiation of monocytes into macrophages, (3) Co-culture of macrophages with K562 leukemic cells and (4) Quantification of phagocytosis by flow cytometry and qualitative analysis by fluorescence microscope.

Innovation

We present a detailed protocol for evaluating tumor phagocytosis by MDMs using pH-sensitive dyes and flow cytometry in 96-well plates. This protocol enables specific detection of phagocytic activity, as pH-sensitive dyes only fluoresce under acidic conditions, indicating successful engulfment and phagolysosomal fusion. Flow cytometry provides efficient and quantitative analysis, even with limited numbers of macrophages and tumor cells. Overall, this protocol offers a straightforward and adaptable approach for investigating phagocytic functions of macrophages, supporting application in cancer immunotherapy research, including the evaluation of macrophage phagocytosis in combination with therapeutic antibodies to enhance antibody-dependent cellular phagocytosis (ADCP), as well as the assessment of bispecific and trispecific killer cell engagers.

Institutional permissions (if applicable)

We isolate human peripheral blood mononuclear cells (PBMCs) from leftover whole blood specimens, collected from leukocyte reduction system (LRS) cones of anonymous healthy donors. For CD14⁺ cell isolation, we obtain approximately 3 mL of blood from each LRS cone. We conduct this study under a protocol approved by the Siriraj Institutional Review Board (SiRB; protocol No. Si 756/2024) in accordance with the Declaration of Helsinki (1975). Each participant receives a detailed information sheet and a comprehensive explanation of the study before signing an informed consent. The recruitment period for this study spans from September 26, 2024, to September 26, 2026.

Isolation of peripheral blood mononuclear cells and sorting of CD14⁺ cells

Timing: 1 day

• 1.
  Isolation of human peripheral blood mononuclear cells (PBMCs) and purification of CD14⁺ cells from PBMCs.

  • a.
    Wipe the external surface of the LRS cone and tubing with 70% ethanol.
  • b.
    Gently invert the LRS cone to mix the contents.
  • c.
    Place the wider side of the LRS cone onto a 50-mL conical tube.
  • d.
    Cut the bottom tubing at the wider side of the cone using sterile scissors, leaving approximately 2 and 3 cm of tubing attached to the cone to facilitate further processing.
  • e.
    Cut the top tubing at the narrower side of the cone to allow the blood product to flow out.
  • f.
    Insert a sterile 200-μL pipette tip into the top tubing, and use a 12-mL syringe to push air through the tip to expel any remaining blood from the cone.
  • g.
    Rinse the LRS cone with sterile PBS.
  • h.
    Add an equal volume of sterile PBS to dilute the blood in the 50-mL conical tube. (e.g., if using 15 mL of blood, add 15 mL of PBS).
  • i.
    Carefully overlay 30 mL of diluted blood onto 9 mL of Lymphoprep™ density gradient medium in a 50-mL conical tube.

    Note: Maintain a Lymphoprep™ to dilute blood ratio of 1:3.

    CRITICAL: Slowly and gently layer the blood using a 10-mL serological pipette. Make sure to avoid mixing the layers.

  • j.
    Centrifuge at 800 × g for 30 min at room temperature (acceleration = 5, brake = 0).

    Note: After centrifugation, distinct layers should appear: plasma, PBMCs (buffy coat), Lymphoprep™ solution, and red blood cells (Figure 1).

  • k.
    Remove the plasma layer down to approximately 0.5 cm above the PBMC layer using a Pasteur pipette.
  • l.
    Carefully collect the PBMC layer (buffy coat) using a Pasteur pipette and transfer it to a new 50-mL conical tube.
  • m.
    Add 15 – 20 mL of sterile PBS to wash the isolated cells.
  • n.
    Centrifuge at 500 × g for 10 min, at room temperature (acceleration = 10, brake = 10) to wash the cells.
  • o.
    Discard the supernatant and repeat steps m-n.
  • p.
    Remove the supernatant and resuspend the cell pellet in 10 mL of RPMI 1640 medium.
  • q.
    Filter the cell suspension through a 40-μm cell strainer.
  • r.
    Perform cell count using trypan blue exclusion assay.
  • s.
    Proceed to the isolation of CD14⁺ cells using the CD14 MicroBeads kit following the manufacturer’s instructions.(https://static.miltenyibiotec.com/asset/150655405641/document_2evdm6e7jp7up842efgqdklc1c/IM0001260.PDF?content-disposition=inline)
  • t.
    Collect CD14⁺ cells in a 15-mL conical tube.
  • u.
    Add 1 mL of RPMI 1640 medium to the tube from step t and centrifuge at 500 × g for 5 min.
  • v.
    Discard supernatant and resuspend the cell pellet in 1 mL of RPMI 1640 medium and count CD14⁺ cells using trypan blue exclusion assay.

    Note: At this stage, CD14⁺ cells can be cryopreserved.

  • w.
    For freezing, resuspend the cell pellet with the pre-chilled freezing medium containing 10% of BloodStor® (DMSO) in 90% fetal bovine serum (FBS).
  • x.
    Aliquot the cell suspension into cryogenic vials at a density of 2×10⁶ viable cells/mL/vial.
  • y.
    Keep the vials in a cryobox at −80°C for overnight and transfer them to a liquid nitrogen tank the next day for long-term storage.
• 2.
  Analysis of sorted cells using flow cytometry.

  • a.
    Prepare 2 × 10⁵ of sorted cells in two 5-mL round-bottom tubes for CD14 and isotype control staining.
  • b.
    Add 1 mL of FACS buffer to each tube and centrifuge at 500 × g for 5 min.
  • c.
    Discard the supernatant and resuspend the cells in 45 μL of FACS buffer containing 5 μL of human AB serum. Incubate at 4°C for 30 min.
  • d.
    Add 0.1 μL of Zombie Aqua (1: 500 dilution) to each tube and mix by vortexing.
  • e.
    Add 1 μL of PE anti-human CD14 antibody and PerCP anti-human CD45 antibody to the staining tube (1: 50 dilution).
  • f.
    Add 1 μL of PE isotype control and PerCP isotype control to the isotype tube (1: 50 dilution).
  • g.
    Incubate both tubes at room temperature for 15 min in the dark.
  • h.
    Add 3 mL of FACS buffer to wash the cells and centrifuge at 500 × g for 5 min.
  • i.
    Discard the supernatant and resuspend the cell pellet in 200 μL of 1% paraformaldehyde (PFA) to fix the cells.
  • j.
    Keep the stained cells at 4°C until performing flow cytometry.

Figure 1.

Isolation of peripheral blood mononuclear cells (PBMCs)

Differentiation of CD14⁺ cells into macrophages and characterization by flow cytometry

Timing: 7 days

• 3.
  Differentiation of CD14⁺ cells into monocyte-derived macrophages (MDMs)

  • a.
    Seed 2.5–3.0 × 10⁶ CD14⁺ cells into a well of 6-well culture plate containing 2 mL of macrophage differentiation medium supplemented with 80 ng/mL M-CSF.
  • b.
    Culture the cells at 37°C with 5% CO₂ for 7 days.
  • c.
    On day 4, change the medium.
  • d.
    On day 7, aspirate the spent medium and wash the well with PBS.
  • e.
    Add 1 mL of TrypLE™ Select Enzyme and incubate for 10 min to dissociate the cells.
  • f.
    Transfer the cell suspension into a 15-mL tube.
  • g.
    Rinse the well with 1 mL of RPMI 1640 culture medium to collect the remaining cells and transfer them to the same tube (step f).
  • h.
    Centrifuge at 500 × g for 5 min.
  • i.
    Discard the supernatant and gently resuspend the cell pellet by tapping.
  • j.
    Add 1 mL of macrophage differentiation medium to the tube.
  • k.
    Count the cells using trypan blue exclusion assay.
  • l.
    Prepare 2 × 10⁵ cells from step k in two 5-mL round-bottom tubes for macrophage markers and isotype control staining.
  • m.
    Add 1 mL of FACS buffer to each tube and centrifuge at 500 × g for 5 min.
  • n.
    Discard the supernatant and resuspend the cells in 45 μL of FACS buffer.
  • o.
    Add 2.5 μL of Human TruStain FcX and incubate at room temperature for 10 min.
  • p.
    Add 2.5 μL of True-Stain Monocyte Blocker and 0.1 μL of Zombie Aqua (1:500 dilution) to each tube, and vortex to mix.
  • q.
    Add 1 μL of APC/Cyanine7 anti-human CD11b antibody, 1 μL of PerCP anti-human CD45 antibody, and 1 μL of PE anti-human CD14 antibody (1: 50 dilution) to the staining tube.
  • r.
    Add 1 μL of APC/Cyanine7 isotype control antibody, PerCP isotype control antibody, and PE isotype control antibody (1: 50 dilution) to the isotype control tube.
  • s.
    Incubate for 15 min at room temperature in the dark.
  • t.
    Add 3 mL of FACS buffer to wash the cells and centrifuge at 500 × g for 5 min.
  • u.
    Discard the supernatant and resuspend the cell pellet in 200 μL of 1% PFA to fix the cells.
  • v.
    Keep the stained cells at 4°C until performing flow cytometry.

Culture of K562 cells (on the same day of isolation of PBMCs)

Timing: 7 days

• 4.
  Thawing of K562.

  • a.
    Quickly thaw frozen K562 cells in a 37°C water bath and immediately transfer the cell suspension into a 15-mL conical tube containing 9 mL of RPMI 1640 basal medium.
  • b.
    Centrifuge at 500 × g for 5 min.
  • c.
    Discard the supernatant and resuspend the cell pellet by tapping.
  • d.
    Add 5 mL of K562 culture medium to the tube, gently mix the cells, and transfer the cells to a 25-cm² tissue culture flask.
  • e.
    Incubate the cells at 37 °C with 5% CO₂ for 3 to 4 days until reaching 90% confluency.
• 5.
  Passaging of K562 cells

  • a.
    When the culture reaches 90% confluency, transfer the cells into a 15-mL conical tube.
  • b.
    Centrifuge at 500 × g for 5 min.
  • c.
    Discard the supernatant.
  • d.
    Resuspend the cell pellet with 1 mL of K562 culture medium.
  • e.
    Perform cell counting using trypan blue exclusion assay.
  • f.
    Seed the cells at a density of 2×10⁵ cells/mL into a 25-cm² tissue culture flask containing 5 mL of K562 culture medium.
  • g.
    Incubate at 37°C with 5 % CO₂ for 3-4 days until reaching 90% confluency.

Note: K562 cells should be maintained within passages 3 to 20.

• 6.
  Cryopreservation of K562 cells.

  • a.
    Prepare a freezing medium for K562 cells and chill it at 4°C for 1 h.
  • b.
    When the culture reaches 90% confluency, transfer the cells into a 15-mL conical tube.
  • c.
    Centrifuge at 500 × g for 5 min.
  • d.
    Discard the supernatant.
  • e.
    Resuspend the cell pellet with the pre-chilled freezing medium.
  • f.
    Aliquot the cell suspension into cryogenic vials at a density of 2×10⁶ viable cells/mL/vial.
  • g.
    Keep the vials in a cryobox at −80°C for overnight and transfer them to a liquid nitrogen tank the next day for long-term storage.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
PE anti-human CD14 antibody (1:50 dilution)	BioLegend	Cat. No. 301806
PE isotype control antibody (1:50 dilution)	BioLegend	Cat. No. 400113
PerCP anti-human CD45 antibody (1:50 dilution)	BioLegend	Cat. No. 368506
PerCP isotype control antibody (1:50 dilution)	BioLegend	Cat. No. 400148
APC/Cyanine7 anti-human CD11b antibody (1:50 dilution)	BioLegend	Cat. No. 101225
APC/Cyanine7 isotype control antibody (1:50 dilution)	BioLegend	Cat. No. 400623
FITC anti-Siglec-9 antibody (1:50 dilution)	BioLegend	Cat. No. 351512
FITC isotype control antibody (1:50 dilution)	BioLegend	Cat. No. 400110
Purified anti-Siglec-9 antibody (1:50 dilution)	BioLegend	Cat. No. 351502
Zombie Aqua™ Fixable viability dye (1:500 dilution)	Biolegend	Cat. No. 423102
Human TruStain FcX (TM) (1:20 dilution)	BioLegend	Cat. No. 422302
True-Stain Monocyte Blocker (1:20 dilution)	BioLegend	Cat. No. 426120
Biological samples
Leukocyte reduction system (LRS) cones	Siriraj Blood Donation Center	–
Chemicals, peptides, and recombinant proteins
Lymphoprep™ density gradient medium	Stemcell™ technology	Cat. No. 07851
RPMI 1640 medium	Gibco™	Cat. No. 31800022
GlutaMax™	ThermoFisher	Cat. No. 35050061
Pen Strep	Gibco™	Cat. No. 15140122
BloodStor® (100% DMSO)	Biolife Solutions®	Cat. No. 410302
Human AB serum	Sigma Aldrich	Cat. No. H4522
TrypLE™ Select Enzyme	Gibco™	Cat. No. 12563011
pHrodo™ iFL Red STP ester	ThermoFisher	Cat. No. P36011
Paraformaldehyde	Sigma Aldrich	Cat. No. P6148
M-CSF	BioLegend	Cat. No. 574806
Critical commercial assays
CD14 MicroBeads kit	Miltenyi Biotec	Cat. No. 130050201
MS column	Miltenyi Biotec	Cat. No. 130042201
MACS™ MultiStand	Miltenyi Biotec	Cat. No. 130042303
MiniMACS™ Separator	Miltenyi Biotec	Cat. No. 130042102
Flow cytometer	BD Biosciences	FACSymphony A1™
Experimental models: Cell lines
K562 cells	ATCC	Cat. No. CCL-243
Software and algorithms
FlowJo v.10	BD Biosciences	N/A
GraphPad Prism v.10	GraphPad	N/A
Other
40-μm cell strainer	SPL Life Science	Cat. No. 93040
5-mL serological pipette	NEST®	Cat. No. 326001
10-mL serological pipette	NEST®	Cat. No. 327001
25-mL serological pipette	NEST®	Cat. No. 328001
Pasteur pipette	NEST®	Cat. No. 318212
15-mL conical tubes	NEST®	Cat. No. 601002
50-mL conical tubes	NEST®	Cat. No. 602002
Cryogenic vial	NEST®	Cat. No. 607001
5-mL round-bottom tubes	SPL Life Science	Cat. No. 40205
Black/clear bottom 96-well tissue culture plates	PerkinElmer	Cat. No. 6055300
Ultra-low attached 96-well plate	Corning®	Cat. No. 7007

Materials and equipment

1× PBS

Reagent	Final concentration	Amount
10× PBS	10×	100 mL
Sterile water	N/A	900 mL
Total	1×	1,000 mL

Store at 4 °C for up to 3 months, pre-warm to 37 °C before use.

FACS buffer

Reagent	Final concentration	Amount
FBS	3%	3 mL
1× PBS	N/A	97 mL
Total	1×	100 mL

Store at 4 °C for up to 3 months.

Macrophage differentiation medium

Reagent	Final concentration	Amount
RPMI 1640	N/A	90 mL
FBS	10%	10 mL
Total	1×	100 mL

Store at 4 °C for up to 1 month, pre-warm to 37 °C before use.

K562 culture medium

Reagent	Final concentration	Amount
RPMI 1640	N/A	88 mL
FBS	10%	10 mL
Pen Strep	1%	1 mL
GlutaMax™	1%	1 mL
Total	N/A	100 mL

Store at 4 °C for up to 1 month, pre-warm to 37 °C before use.

Freezing medium for CD14⁺ cells

Reagent	Final concentration	Amount
FBS	90%	4.5 mL
BloodStor® (100% DMSO)	10%	0.5 mL
Total	N/A	5 mL

Prepare freshly and chill in 4 °C before use.

Freezing medium for K562 cells

Reagent	Final concentration	Amount
K562 culture medium	90%	4.5 mL
BloodStor® (100% DMSO)	10%	0.5 mL
Total	N/A	5 mL

Prepare freshly and chill in 4 °C before use.

Step-by-step method details

Phagocytosis assay

Timing: 1 day and 4 h

We evaluate in vitro tumor phagocytosis assay by co-culturing MDMs with K562 leukemic cells. Before co-culture, we stain K562 cells with pHrodo™ Red, which emits red fluorescence only after engulfment into acidic phagosomes. To enhance phagocytic activity, we treat the co-culture with an anti-Siglec-9 antibody to disrupt inhibitory signaling.

• 1.Differentiate CD14⁺ cells for 7 days at 37°C, 5% CO₂ in 6-well culture plates at a density of 2.0–3.0 × 10⁶ cells/well.
• 2.On day 7, aspirate the spent medium and wash the well with PBS.
• 3.Dissociate the cells with 1 mL of TrypLE™ Select Enzyme and incubate for 10 min at 37°C, 5 % CO₂.
• 4.Transfer the cell suspension into a 15-mL tube.
• 5.Rinse the well with 1 mL of RPMI 1640 culture medium and combine with the same tube (step 4).
• 6.Centrifuge at 500 × g for 5 min.
• 7.Discard the supernatant and resuspend the cell pellet by tapping.
• 8.Add 1 mL of macrophage differentiation medium to the tube.
• 9.Count viable cells using trypan blue exclusion assay.
• 10.Prepare 0.05 μg/mL pHrodo™ iFL Red STP ester by diluting 0.1 μL of 2 mg/mL stock in 4 mL of PBS.
• 11.Stain 2×10⁶ K562 cells with 2 mL of pHrodo™ iFL Red STP ester in a 15-mL conical tube, and incubate for 1 h at 37°C in the dark.
• 12.Wash with 4 mL of RPMI 1640 culture medium containing 10% FBS.
• 13.Centrifuge the cells at 500 × g for 5 min.
• 14.Resuspend the cell pellet with 1 mL of phenol red-free RPMI 1640 medium.
• 15.Count cells using trypan blue exclusion assay.
• 16.Co-culture 5×10⁴ MDMs (step 9) with 5×10⁴, 10×10⁴, 20×10⁴, 30×10⁴ of pHrodo-red labeled K562 cells (step 15) in an ultra-low attached, 96-well plate (E: T ratios = 1: 1, 1: 2, 1: 4, 1: 6), and incubate for 1 h at 37°C.

Note: For anti-Siglec-9 treatment, add 1 μL of anti-Siglec-9 antibody (10 μg/mL) in the co-culture from step 16 (1:50 dilution) and incubate for 1 h at 37°C before adding K562 cells at E:T ratio of 1:1.

• 17.Stop phagocytic reaction by centrifuging the plate at 500 × g for 5 min, 4°C.
• 18.Discard the supernatant by flicking the plate.
• 19.Wash with 100 μL of FACS buffer and centrifuge at 500 × g for 5 min.
• 20.Discard the supernatant by flicking the plate.
• 21.Resuspend cells with 45 μL of FACS buffer and 2.5 μL of Human TruStain FcX.
• 22.Incubate at room temperature for 10 min.
• 23.Add 2.5 μL of True-Stain Monocyte Blocker and 0.1 μL of Zombie Aqua (1:500 dilution).
• 24.Add 1 μL of APC/Cyanine7 anti-human CD11b antibody (1: 50 dilution).
• 25.Incubate at room temperature for 15 min in the dark.
• 26.Add 100 μL of FACS buffer for washing and transfer the cells from each well to a 5-mL round-bottom tube.
• 27.Centrifuge at 500 × g for 5 min.
• 28.Discard the supernatant and resuspend the cell pellet in 200 μL of 1% PFA.
• 29.Store the stained cells at 4°C until performing flow cytometry.

Fluorescence-based phagocytosis imaging

Timing: 1 day 4 h

We perform a parallel assay in a black/clear bottom 96-well plate for microscopic visualization.

• 30.Differentiate CD14⁺ cells at 37°C, 5 % CO₂ for 7 days in a black/clear bottom 96-well plate at a density of 5×10⁴ cells/well.
• 31.Label K562 cells with pHrodo Red as described above in Step 16.
• 32.Co-culture MDMs with the pHrodo-red labeled K562 cells and centrifuge at 50 × g for 50 s to promote contact.
• 33.Incubate at 37°C for 1 h.
• 34.Centrifuge at 500 × g for 5 min, 4°C.
• 35.Discard the supernatant by flicking the 96-well plate.
• 36.Wash with 100 μL of PBS and centrifuge at 500 × g for 5 min.
• 37.Discard the supernatant by flicking the 96-well plate.
• 38.Fix with 200 μL of 1% PFA and incubate at room temperature for 20 min, then centrifuge at 500 × g for 5 min.
• 39.Discard the supernatant by flicking the 96-well plate.
• 40.Wash with 100 μL of PBS and centrifuge at 500 × g for 5 min.
• 41.Discard the supernatant by flicking the 96-well plate.
• 42.Repeat the washing step (steps 39 to 40) twice.
• 43.Add 100 μL of PBS containing Hoechst (1:1000 dilution) and incubate at room temperature for 20 min.
• 44.Acquire images of phagocytic events within macrophages using a fluorescence microscope.

Expected outcomes

Figure 2 illustrates the experimental workflow for assessing the phagocytic activity of monocyte-derived macrophages (MDMs) against K562 leukemic cells. We first isolate PBMCs from LRS cones using density gradient centrifugation, enrich CD14⁺ monocytes by cell sorting, and subsequently differentiate them into macrophages in the presence of 80 ng/mL macrophage colony-stimulating factor (M-CSF). Upon sorting, CD14⁺ monocytes appear small and round (Figure 3A). After 7 days of differentiation, the cells adhere to the plate and exhibit a larger, more elongated morphology (Figure 3A). Flow cytometric analysis confirms the successful isolation of CD14⁺ monocytes through the expression of CD14 and CD45 (Figure 3B). Following differentiation, macrophages express CD14, CD45 and CD11b, CD80, HLA-DR, CD163 and CD206 indicating successful maturation and polarization (Figure 3C).

Figure 2.

Schematic workflow of this study

Figure 3.

Characterization of CD14⁺ monocytes and macrophages

(A) Representative morphology of CD14⁺ monocytes and differentiated macrophages.

(B) Flow cytometric histograms showing marker expression in sorted monocytes, C. Flow cytometric histograms showing marker expression in macrophages.

Following the 7-days differentiation period, we co-culture macrophages with pHrodo Red-labeled K562 cells at varying effect-to-target (E: T) ratios. After 1 h of incubation, we capture representative images and dissociate the co-cultures for staining with macrophage-specific antibodies and subsequent flow cytometric analysis. To distinguish between macrophages from phagocytosed tumor cells, we employ a gating strategy based on side scatter area (SSC-A) versus forward scatter area (FSC-A) to identify and exclude doublets. While a protein-containing buffer is used, the staining intensity was sufficient to reliably distinguish and exclude non-viable cells in Figure 4A. Subsequently, we gate on CD11b (APC/Cyanine 7) to identify macrophage population and pHrodo Red (PE) to detect internalized tumor cells (Figure 4A).¹ We include macrophage-only and tumors-only controls to ensure accurate gating. After optimizing the gating strategy, we apply it to the co-cultured samples to quantify the frequency of phagocytosis within the total macrophage population. We calculate the percentage of phagocytosis using the equation provided in Figure 4B. Our results demonstrate that MDMs phagocytose K562 cells in a dose-dependent manner. Specifically, E: T ratios of 1: 4 and 1: 6 display significant increases in phagocytosis compared to E:T ratio of 1:1 (Figure 4B). Additionally, we observe red fluorescence signals within the macrophages, confirming active phagocytosis (Figure 4C).

Figure 4.

Evaluating phagocytosis of K562 cells by macrophages

(A) Gating strategy used to identify macrophages and internalized tumor cells, (B) Quantification of phagocytic activity (% phagocytosis) at different E: T ratios (Data represent mean ± SD (N = 3–5), ∗∗ p-value < 0.01, ∗∗∗∗ p < 0.0001 and ns indicates no significant difference. Statistical analysis was performed using One-Way ANOVA), (C) Representative images of active phagocytosis of pHrodo Red–labeled K562 cells by macrophages.

Siglec-9 (Sialic acid-binding immunoglobulin-like lectin-9) functions as an inhibitory immune checkpoint that suppresses myeloid cell phagocytosis upon binding to sialoglycan ligands on tumor cells.^2,3,4 Disruption of the siglec-9/ligand interaction effectively removes this “don’t eat me: signal” thereby enhancing the phagocytic clearance of leukemic cells.^5,6,7 To identify the primary target population for this immune checkpoint, we screen Siglec-9 expression across major PBMC subsets. Our screening results reveal that CD14⁺ monocytes exhibit the highest percentage and mean fluorescence intensity (MFI) of Siglec-9 compared with CD4⁺ T cells, CD8⁺ T cells and CD56⁺ NK cells (Figure 5A). Consistent with this observation, treatment with an anti-Siglec-9 blocking antibody significantly enhances macrophage-mediated phagocytosis of K562 cells (Figure 5B).

Figure 5.

Assessing the functions of Siglec-9 on macrophages

(A) Siglec-9 expression on PBMCs (percentage (%) and mean fluorescence intensity (MFI)), (B) Phagocytic activity (%) in the presence of Siglec-9 blockade (10 μg/mL). Data represent mean ± SD (N = 3), ∗ p-value < 0.05. Statistical analysis was performed using One-Way ANOVA.

Overall, this protocol provides a robust platform for evaluating tumor phagocytosis by MDMs using pH-sensitive dyes in a 96-well format. Flow cytometry enables quantitative analysis, allowing objective measurement of phagocytic frequency across thousands of individual cells. This result is complemented by qualitative analysis via fluorescence microscopy, which provides essential visual confirmation that the pH-sensitive signals from within the acidic environment of the phagolysosomes, rather than from extracellular adherent targets. As a result, this protocol provides a straightforward and adaptable approach for investigating phagocytic functions of macrophages, supporting applications in cancer immunotherapy research.

Quantification and statistical analysis

We calculate the percentage of phagocytosis using the Equation (CD11b⁺ pHrodo⁺/CD11b⁺) × 100. We perform statistical comparisons using One-Way ANOVA in GraphPad Prism.

Limitations

Macrophages may engulf apoptotic bodies, membrane fragments, or cellular debris instead of intact leukemic cells. This phenomenon can overestimate the “phagocytic index” as macrophages that internalize only small fragments may still appear positive for the target fluorophore. We utilize Siglec-9 blockade as a model to enhance phagocytosis. While a 1-hour pre-incubation with anti-Siglec-9 antibody is sufficient to observe a significant functional effect in this study, the kinetics of receptor saturation may vary depending on antibody clones and primary donor cells. Furthermore, although our untreated control group displays a clear baseline for phagocytic activity, including isotype controls in future experiments will further help to exclude potential non-specific Fc-receptor engagement.

Troubleshooting

Problem 1

The traditional “flicking” method to remove supernatants often causes the accidental detachment and loss of both macrophages and non-internalized target cells. This loss distorts the final cell counts and produces inaccurate data.

Potential solution

Use a vacuum aspiration to replace the flicking steps.

Problem 2

Phagocytic activity and Siglec-9 expression levels vary among healthy donors due to age, genetics and previous immune exposure. This variability affects the therapeutic effects of Siglec-9 blockade.

Potential solution

Instead of reporting raw percentages, we can calculate the “Phagocytic Fold Change” or “Relative Phagocytosis Index” by comparing the treated group directly with the control from the same donor.

Problem 3

The efficacy of the anti-Siglec-9 treatment depends on the binding affinity of the antibody and its saturation of the surface receptors. If the concentration is too low, the don’t eat me signal remains active. If it is too high, it may cause non-specific binding or steric hindrance.

Potential solution

Perform an antibody titration assay (dose-response curve) to determine the saturating concentration required for effective blockade before initiating co-culture with K562 cells.

Problem 4

APC/Cy7 for CD11b (macrophage marker) can exhibit signal spread into adjacent channels, including the PE channel used for pHrodo Red if compensation is not correctly configured. This spectral spillover may lead to false-positive results.

Potential solution

Always include single-stain controls (e.g., macrophage stained with anti-CD11b-APC/Cy7 only and K562 stained with pHrodo Red only) to generate an accurate compensation matrix before sample acquisition.

Problem 5

Mechanical scraping of adherent macrophages can cause cell damage and increase autofluorescence, resulting in high background signals.

Potential solution

Use a gentle dissociation reagent, such as TrypLE™ Select Enzyme, facilitating the release of adherent cells while maintaining viability and minimizing background noise.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Methichit Wattanapanitch (methichit.wat@mahidol.ac.th).

Technical contact

For technical specifics on executing the protocol, Dr. Saitong Muneekaew (saitong.mun@mahidol.ac.th) will provide support to ensure its correct implementation.

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate new data or code.

Acknowledgments

The authors thank the Siriraj Core Facilities and Research Support Service and the Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University. This research is funded by the Siriraj Research Fund, Faculty of Medicine Siriraj Hospital, Mahidol University (R016733022 to S.M. and IO R016837001 to M.W.); the National Research Council of Thailand (NRCT); and the Faculty of Medicine Siriraj Hospital, Mahidol University through the Mid-Career Researcher Grant (N42A660313 to M.W.). The graphical abstract/figures were created using Biorender.com.

Author contributions

Conceptualization, S.M. and M.W.; methodology, data analysis, and validation, S.M. and M.W.; investigation, S.M., T.N., P.S., K.S., and K.R.; data curation, S.M. and M.W.; writing – original draft preparation, S.M.; writing – review and editing, S.M., T.N., P.S., and M.W.; funding acquisition, M.W. All authors have read and agreed to the published version of the manuscript.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used Gemini and ChatGPT to improve the clarity and quality of the English language. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.