Protocol for monitoring phagocytosis of cancer cells by TAM-like macrophages using imaging cytometry

Summary

Here, we present a protocol for monitoring phagocytosis by M2-type macrophages using automated counting of phagocytic events with an imaging cytometer. We describe steps for isolating and differentiating peripheral blood mononuclear cell (PBMC)-derived monocytes into M2-like macrophages, preparing cancer cells expressing a green fluorescence marker, labeling with a pH-sensitive dye, and co-culturing with macrophages. We then outline procedures for enumerating phagocytic events using an imaging cytometer.

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

Graphical abstract

Highlights

• •Protocol for differentiating PBMC-derived monocytes into M2-like macrophages
• •Protocol for labeling cancer cells to track phagocytosis
• •Steps for using an imaging cytometer to count phagocytic events in 96-well plate format

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

Here, we present a protocol for monitoring phagocytosis by M2-type macrophages using automated counting of phagocytic events with an imaging cytometer. We describe steps for isolating and differentiating peripheral blood mononuclear cell (PBMC)-derived monocytes into M2-like macrophages, preparing cancer cells expressing a green fluorescence marker, labeling with a pH-sensitive dye, and co-culturing with macrophages. We then outline procedures for enumerating phagocytic events using an imaging cytometer.

Before you begin

Phagocytosis, a crucial function of the innate immune system, involves macrophages engulfing and digesting cellular debris, pathogens, and cancer cells.^2,3,4 Tumor-associated macrophages (TAMs) are a specialized subset within the tumor microenvironment that can be polarized into M1 or M2 types. M1 macrophages exhibit pro-inflammatory and anti-tumor properties, while M2 macrophages support tumor growth and metastasis through anti-inflammatory responses.^5,6,7 Understanding TAM-mediated phagocytosis is essential as it acts as an innate immune checkpoint, influencing the immune system’s ability to combat cancer. M2 macrophages are particularly relevant in cancer because they secrete anti-inflammatory cytokines and growth factors (e.g., IL-10 and TGF-β), that promote tumor progression and suppress effective immune responses.^5,8 Investigating the mechanisms behind M2 polarization and its impact on phagocytosis is crucial for developing effective immunotherapeutic strategies.

This protocol presents an optimized approach for monitoring phagocytosis of ovarian cancer cells by M2-differentiated (TAM like) macrophages and, provides a valuable tool for studying the complex interactions between phagocytes and cancer cells. This protocol outlines the steps for monitoring the events of phagocytosis using a Celigo Image Cytometer. We describe the specific steps for evaluating phagocytosis induced by small-molecule inhibitors or biologics in ovarian cancer cells, which can be applied for other cell types and models.

Institutional permissions

This protocol requires institutional approval for handling biohazardous agents. The use of recombinant lentiviral vectors and human blood was approved as per the guidelines of the Institutional Biosafety Committee and Institutional Regulatory Board.

Preparation of macrophages - Isolation of peripheral blood mononuclear cells (PBMCs)

Timing: 2 h

The steps below describe the process of PBMC isolation from Leukopaks.

Note: Leukopaks offer high yield and ideal for efficient PBMC isolation, particularly for large-scale studies. However, the following steps of PBMC isolation are also applicable for human donor-derived peripheral blood samples or cord blood samples.

Note: Use leukopaks/blood samples as soon as they are obtained. Storing them at 4°C for an extended period may affect the viability of PBMCs and the monocytes.

• 1.Carefully transfer 10 mL of blood sample into a sterile 50 mL conical tube.

Note: Perform all the procedure in a biosafety cabinet.

• 2.Dilute the blood with an equal volume of sterile 1X PBS. For example, mix 10 mL of blood with 10 mL of PBS to make a total volume of 20 mL.
• 3.Add 15 mL of Ficoll-Paque to a new 50 mL conical tube.
• 4.Gently layer the diluted blood on top of the Ficoll-Paque.
• 5.To layer, tilt the tube and slowly pipette the blood-PBS mixture down the side of the tube to form a distinct layer above the Ficoll-Paque.

CRITICAL: Avoid mixing the blood with the Ficoll-Paque.

• 6.Centrifuge the tube at 400 × g for 30 min at 4°C in the swing bucket rotor.

CRITICAL: Make sure the centrifuge brake is turned off to prevent disruption of the gradient. Do not use a fixed angle rotor.

• 7.After centrifugation, distinct layers are observed: plasma at the top, a white cloudy layer (buffy coat) containing PBMCs, the Ficoll-Paque layer, and erythrocytes and granulocytes at the bottom.
• 8.Using a sterile pipette, carefully aspirate the upper layer containing plasma and platelets without disturbing the buffy coat layer.
• 9.Carefully aspirate the buffy coat layer using a sterile pipette and transfer it to a new 50 mL conical tube.

CRITICAL: Avoid aspirating Ficoll-Paque and the other cell layers.

• 10.Add three volumes of sterile PBS to the PBMCs (e.g., if you have 5 mL of PBMCs, add 15 mL of PBS).
• 11.Centrifuge at 300 × g for 10 min at room temperature.
• 12.Carefully aspirate the supernatant without disturbing the pellet.
• 13.Add 1 mL RBC lysis buffer to the cell pellet and gently resuspend.
• 14.Incubate for 5–10 min at room temperature.
• 15.After incubation add an excess of cold PBS to stop the lysis reaction.
• 16.Centrifuge at 300–400 g for 5–10 min at 4°C to pellet PBMCs.
• 17.Wash PBMCs twice with cold PBS to remove any residual lysis buffer.
• 18.Resuspend the final PBMC pellet in an appropriate volume of RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin.
• 19.Count the cells using a hemocytometer or an automated cell counter.
• 20.Determine the yield and viability of the isolated PBMCs using the trypan blue staining.
• 21.Use the isolated PBMCs immediately for downstream applications or cryopreserve them in a freezing medium (10% DMSO in FBS) for future use.

Pause point: PBMCs can be cryopreserved at this stage for later use.

• 22.For cryopreservation aliquot the cells into cryovials.
• 23.Place them in a controlled-rate freezer or an isopropanol freezing container at −80°C overnight.
• 24.Transfer the cells to liquid nitrogen for long-term storage.

Preparation of target cancer cells - GFP tagging

Timing: 2 weeks

• 25.Use the lentiviral-based expression system for stable expression of GFP.
• 26.Transduce cancer cells at 30%–50% confluency with lentivirus-containing GFP transgene.
• 27.Incubate for 24–48 h, confirm GFP expression using fluorescence microscopy, and proceed with cell culture.
• 28.Maintain GFP-tagged cells under appropriate conditions for long-term expression of fluorescent marker.

Note: The quantification of phagocytic events in macrophages (red, fluorescent foci counting) by Celigo Image Cytometer does not necessarily require the GFP tagging. However, for visualization of phagocytic events by the cytometer, fluorescence microscopy or analysis by flow cytometry require fluorescent labeling of cancer cells.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
APC anti-mouse/human CD11b (M1/70) (dilution: 1:50)	BioLegend	Cat# 101212 RRID: AB_312795
PE anti-human CD206 (15–2) (dilution: 1:50)	BioLegend	Cat# 321118 RRID: AB_571911
APC human IgG1 isotype (QA16A12) (dilution: 1:50)	BioLegend	Cat# 403506 RRID: AB_2942002
PE human IgG1 isotype (QA16A12) (dilution: 1:50)	BioLegend	Cat# 403504 RRID: AB_3097044
Human TruStain FcX (dilution: 1:100)	BioLegend	Cat# 422302 RRID: AB_2818986
Anti-human CD24 (SN3) (unconjugated) (10 μg/mL)	Novus Bio	Cat# NB100-64861 RRID: N/A
Anti-human CD47(CC2C6) (unconjugated) (10 μg/mL)	BioLegend	Cat# 323102 RRID: AB_756132
Human IgG1 isotype control (unconjugated) (10 μg/mL)	BioXCell	Cat# BE0297
Biological samples
Leukopaks	Rhode Island Blood Center (Providence, RI)	N/A
Chemicals, peptides, and recombinant proteins
RPMI 1640 medium	Thermo Fisher Scientific	Cat# 61870036
DMEM with high glucose	HyClone	Cat# SH30022.01
Fetal bovine serum (FBS)	R&D Systems	Cat# S11550
Sodium pyruvate	Thermo Fisher Scientific	Cat# 11360070
Monocyte attachment medium (MAM)	MilliporeSigma	Cat# C-28051
Non-essential amino acids	Thermo Fisher Scientific	Cat# 11140050
L-glutamine	Thermo Fisher Scientific	Cat# A2916801
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	Cat# 15140122
TrypLE Express enzyme	Thermo Fisher Scientific	Cat# 12604013
Ficoll Paque Plus	MilliporeSigma	Cat# GE17-1440-02
1X RBC lysis buffer	eBioscience	Cat# 00-4333-57
Recombinant human M-CSF	PeproTech	Cat# AF-300-25
Recombinant human IL-4	PeproTech	Cat# 200-04
Recombinant human IL-13	PeproTech	Cat# 200-13
Bovine serum albumin	MilliporeSigma	Cat# A3294
Bestatin	SelleckChem	Cat# S1591
Tosedostat	MedChemExpress	Cat# HY-14807
Docetaxel	SelleckChem	Cat# S1148
DMSO	MilliporeSigma	Cat# D8418
Sodium azide	MilliporeSigma	Cat# S2002
EDTA	MilliporeSigma
DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride)	Thermo Fisher Scientific	Cat# D1306
pHrodoRed, SE	Invitrogen	Cat# P36600
Trypan blue	Thermo Fisher Scientific	Cat# 15250061
Critical commercial assays
Universal Mycoplasma detection kit	ATCC	Cat# 30–1012K
Experimental models: Cell lines
OVCAR8	Creative Biolabs	Cat#IOC-ZP305
HEK-293T	ATCC	CRL-3216
Software and algorithms
FlowJo v10	FlowJo, LLC	RRID: SCR_008520
BioRender	BioRender	https://www.biorender.com/
Prism 9.0	GraphPad	https://www.graphpad.com/
Other
Celigo image cytometer	Nexcelom Bioscience	N/A
Flow cytometer	Bio-Rad ZE5 cell analyzer	N/A
96-Well plates	Corning	Cat# 353072
Conical tubes (15 mL, 50 mL)	Corning	Cat# 352095 Cat# 352070
Cell counter	Invitrogen Countess 3 FL automated cell counter	Cat# AMQAF2000

Materials and equipment

Macrophage differentiation medium

Reagent	Final concentration	Amount
Glutamine-free RPMI-1640 medium	N/A	440 mL
Fetal Bovine Serum (FBS)	10%	50 mL
Penicillin-Streptomycin	100 U mL–1 /100 mg mL–1	0.5 mL
MEM non-essential amino acids	1X	5 mL
L-glutamine	2 mM	5 mL
Sodium pyruvate	1 mM	1 mL
M-CSF	20 ng mL–1	N/A
Total	N/A	500 mL

Note: Store at 4°C for up to 2 weeks. M-CSF stock should be stored at −20°C and added at the time of experiment.

FACS buffer

Reagent	Final concentration	Amount
Phosphate-buffered saline (PBS)	1X	10 mL of 10X stock PBS
Bovine serum albumin (BSA)	1% (w/v)	1 g
EDTA (Ethylenediaminetetraacetic acid)	2 mM	58.45 mg
Sodium azide	0.01% w/v	0.1 mg
Total	N/A	100 mL

Note: Store at 4°C for up to 1 month. Check for contaminant growth prior to use.

Step-by-step method details

Isolation and differentiation of monocytes

Timing: 7 days

This section describes the process of monocyte isolation and differentiation in M2 like macrophages.

• 1.Seed the PBMCs into 10 cm dishes at a density of 1×10⁶ cells per dish in Monocyte Attachment Medium (MAM) supplemented with 10% fetal bovine serum (FBS) and 1x penicillin/streptomycin.
• 2.Incubate the dishes for 1 h at 37°C in CO₂ incubator.
• 3.After incubation, carefully remove the medium along with the non-adherent cells.
• 4.Gently wash the cells twice with RPMI medium or PBS to eliminate the non-adherent cells.
• 5.Add the medium to the side of the dish with a gentle rocking motion and then remove the medium using an aspirator.

Note: Avoid flushing the medium directly onto the plate surface to prevent dislodging the loosely attached monocytes.

Alternatives: Monocyte Attachment Medium contains all the growth factors and supplements necessary for providing optimal cell health and efficient adherence selection of monocytes from freshly isolated mononuclear cells. Alternatively, monocytes can be isolated by negative selection method using Human Monocyte Isolation Kit as per the manufacturer’s instructions (https://cdn.stemcell.com/media/files/pis/10000011612-PIS_00.pdf).

Optional: Check the purity of isolated monocytes by flow cytometry using monocyte-specific markers using macrophage-specific markers such as CD11b.

• 6.Prepare 500 mL of macrophage differentiation medium by adding recombinant human M-CSF at final concentration of 20 ng/mL to the complete RPMI medium.
• 7.Mix thoroughly to ensure even distribution of M-CSF in the medium.
• 8.Seed PBMC-derived monocytes at a density of 1×10⁶ cells per T75 flask in 20 mL of macrophage differentiation medium.
• 9.Allow the monocytes to differentiate over a period of 7 days by incubating at 37°C with 5% CO_2.
• 10.Every 2 days, carefully aspirate the old medium and replace it with fresh medium supplemented with M-CSF to maintain a final concentration of 20 ng/mL.

CRITICAL: Consistently maintain M-CSF supplementation in the medium to ensure proper differentiation.

• 11.Monitor the morphological changes such as increased cell size enhanced adherence to the flask surface, indicative of macrophage differentiation.

Note: Well-differentiated macrophages exhibit a spread-out, amoeboid shape with prominent pseudopodia (extensions), granular cytoplasm, and uniform size and shape

Induction of M2-type phenotype

Timing: 3 days

This step described the polarization of the macrophages into M2-type.

• 12.Carefully aspirate the medium from the macrophage culture dish.
• 13.Add 10 mL RPMI media containing M-CSF (20 ng/mL) and IL-4 and or IL-13 at a final concentration of 20 ng/mL.
• 14.Incubate the cells for an additional 72 h at 37°C with 5% CO₂.

Analysis of M2 polarized macrophages by flow cytometry

Timing: 2 h

This step described the harvesting of polarized macrophages for downstream application and evaluation of the successful polarization.

• 15.After 3–4 days of incubation with IL-4 and IL-13, carefully remove the medium and gently wash the cells with PBS.
• 16.
  Use non-enzymatic cold shock method to detach the macrophages.

  Note: Detaching macrophages from a cell culture plate is challenging due to their strong adherence to the substrate. Trypsinization and scraping of cells can damage cell surface receptors and compromise cell viability. Therefore, the cold shock method is recommended for preparing macrophages for phagocytosis assay.

  • a.
    Carefully aspirate the culture media from the macrophage culture dish without disturbing the cells.
  • b.
    Gently rinse the cells with PBS.
  • c.
    Incubate the monolayer with 5 mM EDTA in PBS at 37°C for 5 min.

    Note: EDTA chelates calcium ions and weakens cell adhesion

  • d.
    Aspirate the EDTA solution from the culture without disturbing the macrophage monolayer.
  • e.
    Add 5 mL cold PBS.
  • f.
    Swirl the PBS around the dish to ensure all cells are exposed to the cold temperature.
  • g.
    Leave the dish at 4°C for 10–15 min.
  • h.
    After cold incubation, gently pipette the PBS up and down 3–5 times to dislodge the cells.

    Note: Hold the pipette at an angle (about 45 degrees) to gently dislodge the cells from the plate, reducing mechanical stress and preserving their morphology. If cells are not adequately detached, pipetting can be repeated. However, excessive mechanical stress and potential damage to the cells should be avoided. Typically, 70%–90% of the cells should detach using the cold shock method.

• 17.Centrifuge the cells at 300 × g for 5 min to pellet them.
• 18.Carefully remove the supernatant and resuspend the cell pellet in PBS.
• 19.Centrifuge again and remove the PBS to ensure a clean cell pellet.
• 20.Resuspend the cells in FACS buffer to achieve a concentration suitable for flow cytometry analysis.
• 21.Divide the cell suspension into separate tubes (100 μL each tube) for each condition to be analyzed.
• 22.Incubate the cells in Fc block (5 μL/million cells) for 10 min at room temperature to block nonspecific antibody binding.
• 23.Include a tube with cells stained with the isotype control antibody to account for non-specific binding.

CRITICAL: Human Fc receptors (FcRs) are highly expressed immune cells. Therefore, blocking of Fc-receptors is crucial for avoiding non-specific signals.

• 24.Add the fluorescently labeled anti-CD11b-APC and anti-CD206-PE antibodies to each tube in 1:100 dilution.
• 25.Incubate the tubes in the dark at 4°C for 30 min. This allows the antibody to bind on the cell surface markers.
• 26.After the incubation, add FACS buffer to each tube to wash away unbound antibodies.
• 27.Resuspend the cells in the 0.5 mL FACS buffer and transfer them to flow cytometry tubes compatible with the instrument.
• 28.Add 0.1 μg/mL DAPI solution for excluding the dead cell population.

CRITICAL: Use only the swinging bucket rotor at speed 300 × g for 5 min to ensure that cells are not damaged.

• 29.Analyze the samples using a flow cytometer.

Note: Be sure to follow proper flow cytometry practices and biosafety guidelines while working with cells and antibodies. Use appropriate controls to adjust compensation and gating settings.

Drug treatment of cancer cells

Timing: 3 days

This step describes the process of treating cultured ovarian cancer cells with the small molecule drug(s) to study its effects on phagocytosis by M2 type macrophages.

• 30.Seed 0.5 × 10⁶ GFP expressing OVCAR8 cells in a 6 well plate and incubate the cells at 37°C in a 5% CO₂ humidified incubator for 12 h to achieve 60%–70% confluency.
• 31.Prepare the drug solution at the desired concentration in RPMI medium.

Note: Always prepare fresh drug solutions to maintain efficacy.

• 32.Remove the existing medium from the cells.
• 33.Add the prepared drug containing medium to the culture dish containing the cancer cells.
• 34.Incubate the cells with the drug solution for 72 h under standard culture conditions (37°C, 5% CO₂).
• 35.After 72 h, remove the drug-containing medium.
• 36.Gently wash the cells with fresh culture medium to remove any residual drug.
• 37.Dissociate the cells by adding 0.5 mL of TrypLE Express and incubating for 5 min.
• 38.Harvest the cells and wash with 1X PBS and prepare cells for subsequent steps.

CRITICAL: Ensuring that the cell lines used in the assay are free of mycoplasma contamination is essential, as the presence of mycoplasma can lead to false signals and compromise the accuracy of the results.

Note: Dissociation with TrypLE express enzyme maintains cell health and preserves expression of surface markers.

Labeling of cancer cells with pHrodoRed dye

Timing: 30 min

This step describes the procedure of efficient pHrodoRed labeling of the cancer cells.

Note: pHrodoRed is a pH-sensitive dye that fluoresces brightly red when exposed to acidic environments, such as the phagolysosomes of macrophages.⁹

Note: pHrodoRed labeling of target cancer cells should be performed simultaneously while harvesting the differentiated macrophages for co-culturing.

• 39.Reconstitute the 1 mg lyophilized pHrodoRed dye in 1 mL DMSO and store the stock solution at −20°C.

Note: Ensure working in a dark environment.

• 40.Wash cancer cells with 1X PBS to remove any residual media components.
• 41.Add the reconstituted pHrodoRed solution at a concentration of 1:20,000 in PBS.

Note: Mix the dye solution well to ensure the even distribution of the dye with cell suspension.

• 42.Incubate the cells with the pHrodoRed dye for 30 min in ice.

CRITICAL: Use only freshly diluted pHrodoRed dye solution. While the stock solution can be stored at −20°C, always dilute the required concentration of dye immediately before use.

• 43.Wash labeled cells at least twice with RPMI supplemented with 10% FBS and 1% penicillin/streptomycin to remove excess dye.

CRITICAL: Thorough washing of the cells is crucial to avoid nonspecific signals from engulfment of cell-free dye particles by macrophage

• 44.Resuspend the cell pellet in an appropriate volume of RPMI medium. Keep the tubes on ice until use.

Alternatives: As an alternative to pHrodoRed dye, other pH-sensitive dyes like pHrodo-Green and CypHer5 can be employed for similar purposes.

Phagocytosis assay

Timing: 6 h

This step describes the setting up of macrophage-cancer cell co-culture plate and reading phagocytic events.

• 45.Harvest the differentiated macrophages from the culture plates as described in the previous step.
• 46.Seed 5 × 10⁴ macrophages per well in low attachment flat-bottom 96-well plates and allow them to settle on the bottom for 2 h.

CRITICAL: Ensure that the macrophages are evenly spread in the well and not in clumps. Clumping can be minimized by gently pipetting the macrophage suspension up and down several times while seeding. Additionally, pass the macrophage suspension through a 70 μm cell strainer to break up any clumps before seeding into a 96-well plate. This ensures uniform distribution and accurate phagocytosis assessment.

CRITICAL: To minimize the edge effect while reading the 96-well plate in the cytometer, avoid using wells located in the outermost rows and columns.

• 47.Once the macrophages are settled, gently aspirate the medium from the wells, without disturbing the macrophages.
• 48.Seed pHrodoRed-labeled cancer cells (5 × 10⁴ per well) in RPMI medium, ensuring the total volume does not exceed 150 μL.

CRITICAL: Centrifuge the co-culture briefly at 100 × g for 2 min to promote the settlement of cancer cells with macrophages into the same plane.

Optional: Add an appropriate amount of opsonizing or immune checkpoint inhibitor antibodies as per experimental requirement.

• 49.Include controls such as target cells without macrophages and untreated co-cultures to validate specificity and baseline phagocytosis respectively.
• 50.Incubate the co-culture for 4 h, allowing sufficient time for the macrophages to engulf the cancer cell.

CRITICAL: Do not incubate the co-culture for more than 12 h, as the proliferation of cancer cells or the death of macrophages may alter the effector-target ratio of the co-culture among treated vs untreated groups.

Optional: Cells can be fixed by adding 4% paraformaldehyde solution to halt the phagocytic process if the instrument is not immediately available for reading the plate.

• 51.Read the phagocytosis events as the number of pHrodoRed⁺ foci per well using a Celigo Image Cytometer (as described below).

Instrument setup

Timing: 15 min

Note: Celigo cytometer is a high-throughput imaging cytometer that allows the visualization and quantification of cells or any florescent entity. It is widely used for various cell-based assays.^10,11 It features a transmission and epifluorescence optical setup with one bright-field (BF) and four fluorescence (FL) imaging channels Blue (Ex/Em: 377/470 nm), Green (Ex/Em: 483/536 nm), Red (Ex/Em: 531/629 nm), and Far Red (Ex/Em: 628/688 nm) using high-power LEDs. Its advanced optics can quickly capture uniform images of 96-well plates in 10–15 min in a bright field and can autofocus based on image contrast or bottom surface thickness.

Note: The Celigo software provides various applications for analyzing fluorescent entities. For quantitative measurement of phagocytosis, the “High Throughput Foci Formation Counting” (Celigo assay ID 06_0001) application can be used. This application involves selecting channels as targets (1, 2, ..., n) depending on the fluorescent markers used in the assay. For a phagocytic assay, the required channels include "Target 1 (bright field) + Target 2 (Green) + Target 3 (Red). The bright-field channel visualizes and counts the total number of cells in each well, while the green and red channels specifically visualize and count GFP-labeled cancer cells and pHrodoRed+ events, respectively.

Note: The bright-field count provides the total cell count per well, ensuring that an equal number of cells were added to each well.

Measurement of phagocytic events by celigo image cytometer

Timing: 30 min

The step describes procedure of instrument set up and acquisition of data (Figure 1A).

• 52.Load the sample plates (co-culture plates) into the instrument and use preset SCAN and ANALYZE settings.
• 53.Select the application; High Throughput Foci Formation Counting and Target 1 + 2+3.
• 54.Select the channels as bright field, red and green.
• 55.Set up the analysis parameter for bright field only and follow the same setup for the fluorescence channels.
• 56.Select hardware-based autofocus to focus cells on the bright field.
• 57.Select the sample wells and start scan.
• 58.After image acquisition, use the preset ANALYZE parameters to first identify the bright field cells as masks then apply them to the green and red fluorescence channels to measure the mean fluorescence foci for each sample.
• 59.Generate the graphic overlay to visualize the counts.

Note: Graphic overlays can highlight fluorescence foci, making them more visible and easier to distinguish from the background (Figure 1B).

• 60.Export the data in CSV file format (see Figure 2A for example plate wise data).
• 61.Analyze the relative difference in the phagocytic events detected in the treatment wells and the control wells.
• 62.
  For analysis.

  • a.
    Calculate the normalized mean of pHrodoRed+ events per well.
  • b.
    Subtract the mean pHrodoRed+ counts/wells with only cancer cells (non-specific counts) from the mean pHrodoRed+ counts in experimental wells (cancer cells and macrophages).
  • c.
    Calculate the normalized phagocytosis or the percent phagocytosis using the formulae as described below (Figure 2B).
  • d.
    To obtain statistical significance, perform the experiment in at least three independent biological replicates.

Alternatives: As an alternative to Celigo imaging cytometer the monitoring of phagocytosis can also be achieved using Incucyte system.

Figure 1.

Celigo Instrument setup and workflow

(A) Flowchart depicting steps involved in running the scan on Celigo cytometer.

(B) Plate-wise and well-wise visualization of data. The total red fluorescence foci count of each well is depicted. The inset displays a magnified view of the graphic overlays, showing red, fluorescent foci representing phagocytic events and green overlay indicating GFP-tagged cancer cells.

Figure 2.

Data collection from Celigo cytometer and calculation of phagocytic index

(A) An example of representative well-wise data generated by Celigo Image Cytometer. Upper panel represent total cell counts in each well and lower panel represents pHrodoRed⁺ foci count in each well.

(B) Formulae that are used to calculate the normalized phagocytosis or the precent phagocytic events. Abbreviations: DMSO; Dimethylsulfoxide, BES; Bestatin, TOS; Tosedostat, DTX; Docetaxel, DMSO; Dimethylsulfoxide, IgG; Immunoglobulin G. Unlabeled cells; unlabeled cancer cells without macrophages. Labeled cells; pHrodo labeled cancer without macrophages.

Expected outcomes

Differentiation of macrophages

Successful differentiation of monocytes into macrophages with high purity can be confirmed by flow cytometry using macrophage-specific markers (CD14, CD45, and CD11b). Differentiated macrophages should display characteristic morphology changes, including an elongated or spindle-like shape compared to the round shape of undifferentiated macrophages (Figure 3).

Figure 3.

Monocytes differentiation into macrophages

(A) Microscopic bright-field image showing day 1, monocytes after 24 h of seeding.

(B) Differentiated macrophages at day 7.

Polarization of macrophages

The M2 polarization of macrophages can be confirmed by assessing the expression of classical M2 markers, such as CD206 and CD163. Macrophages treated with the cytokines, IL-4 and IL-13 exhibit increased expression of CD206. This can be monitored through surface staining and flow cytometric analysis of the treated cells. Successful differentiation into the M2 phenotype should be evidenced by the expression of these markers, confirmed via flow cytometry or immunostaining (Figure 4).

Figure 4.

Confirmation of macrophage polarization to M2 type

(A) Gating strategy of immunostaining of M2 differentiated macrophages, DAPI⁻, CD11b-APC, CD206-PE.

(B) Flow cytometry plots (left) and quantitation (right), showing percentage of CD206+ macrophages after treatment with interleukins, IL-4 and/or IL-13 for 3 days.

Measurement of phagocytosis induced by small-molecule inhibitors or MAbs

Small molecule inhibitors that interfere with the surface presentation of the immune checkpoint protein CD24, such as bestatin and tosedostat,¹ or the chemotherapeutic drug docetaxel, which induces surface localization of prophagocytic signals like calreticulin (CALR), are expected to increase phagocytosis of ovarian cancer cells.¹ The protocol described here enables the monitoring of phagocytosis affected by multiple agents in a single plate, along with positive and negative controls. This setup allows for medium-throughput analysis of cellular phagocytosis modulators by M2 polarized macrophages, representing the tumor-associated macrophage (TAM) phenotype.

In this representative experiment, GFP-labeled OVCAR8 cells were pretreated with bestatin (20 μM), tosedostat (5 μM) and docetaxel (100 nM) or DMSO control and co-cultured with M2 polarized human PBMC-derived macrophages. For positive control, immune checkpoint inhibitor antibodies targeting CD24 and CD47, along with antibody control IgG, were included. The co-culture was set up in a Celigo-compatible clear and flat-bottom 96-well plate. In the designated wells of the co-culture plate, 10 μg/mL of each IgG (antibody control), anti-CD24, and anti-CD47 antibodies were added. As shown in Figure 5, drug treatments or the treatment with CD24 and CD47 antibodies led to a significant increase in phagocytosis compared to DMSO or IgG controls. The experiment was performed in three biological replicates, with each biological replicate consisting of at least six technical replicate wells from each group.

Figure 5.

Phagocytosis of ovarian cancer cells by small molecule drugs and immune checkpoint inhibitors

Plot showing normalized phagocytosis of ovarian cancer cells induced by small molecule drugs and immune checkpoint inhibitor monoclonal antibodies (anti-CD24 or anti-CD47). Data was normalized with respect to DMSO or an isotype (IgG) antibody control. Data are presented as mean ± SD from three independent experiments. p values were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Quantification and statistical analysis

• 1.Ensure robustness by compiling data from multiple wells and conducting experiments in biological replicates.
• 2.Perform statistical analysis (e.g., t-tests, ANOVA) to compare phagocytic activity under different experimental conditions.
• 3.Generate graphs and plots to visualize the phagocytic index and percentage of phagocytic macrophages.

Limitations

The protocol is resource-intensive, requiring specialized equipment and reagents, and involves ethical considerations, particularly when using primary cells from donors. Fluorescent labeling and imaging techniques are prone to artifacts such as non-specific binding, autofluorescence, or crosstalk between different fluorescent channels, potentially confounding the interpretation of results. Additionally, the protocol does not provide sufficient information on whether macrophages have phagocytosed entire cancer cells or merely parts of a pH-sensitive dye-labeled fragment from dying cancer cells. Validation of the results by confocal imaging or flow cytometry is recommended. Furthermore, the protocol relies on a high-throughput imaging cytometer, which may not be readily available in all research laboratories due to its cost and the need for specialized training to operate. Consequently, careful experimental design and meticulous execution are essential to mitigate these limitations and obtain reliable data.

Expanded applications

This phagocytosis protocol has broad applications across multiple research areas. It provides insights into immune cell functions and cytokine effects on phagocytosis and phagocytosis-mediated immune evasion mechanisms. The protocol can be used to investigate the impact of gene alterations on phagocytic activity, explore tumor microenvironment interactions and assess immunotherapy effects. Additionally, the 96-well plate-based format enables high throughput screening of drugs/small-molecules compounds or biologics that modulate phagocytosis.

Troubleshooting

Problem 1

Poor differentiation of primary monocytes into macrophages (Figure 6).

Figure 6.

Assessing proper differentiation of human monocytes to macrophages in vitro

Differentiation of monocytes to macrophages can be assessed by monitoring morphological features using microscopy. Properly differentiated macrophages exhibit a spread-out, amoeboid shape with prominent pseudopodia (extensions), granular cytoplasm, and uniform size and shape. Conversely, poorly differentiated cells appear round, small, and less spread out, with varying shapes and sizes indicating a mixed population of differentiated and undifferentiated cells. Microscopic bright-field image of Day 7 showing poor (A) or good (B) differentiation of macrophages.

Potential solution

Ensure that the differentiation medium contains the necessary growth factors (such as M-CSF or GM-CSF) at optimal concentrations. Monitor the differentiation process closely and adjust the duration of differentiation if needed, based on cellular morphology. Replenish the media with growth factors regularly.

Problem 2

Failure to polarize macrophages into M2-type.

Potential solution

Ensure that polarization cytokines, such as IL-4 and IL-13, are used at optimal concentrations and for the recommended incubation periods. Verify both the activity and expiration dates of the cytokines and other reagents to ensure their effectiveness. Additionally, include appropriate controls, such as M1 macrophage polarization using IFN-γ and LPS, to confirm the functionality of the polarization protocol.

Problem 3

High background fluorescence.

Potential solution

Use only freshly diluted pHrodoRed dye and adhere strictly to the recommended concentration and incubation time for optimal results. It is essential to implement proper controls to distinguish between specific signals and background noise. After labeling cancer cells with pHrodoRed dye, ensure that they are thoroughly washed to remove any unbound dye, as residual dye can increase background fluorescence and interfere with final observations. Additionally, optimize the imaging settings by adjusting exposure time and gain to minimize background fluorescence and enhance the clarity of your results.

Problem 4

Variability in phagocytosis assay results.

Potential solution

Standardize the experimental protocol by ensuring consistency in the source and preparation of macrophages. Maintain uniform cell density and consistent phagocytic target-to-cell ratios across all experiments. Conduct time-course studies to determine the optimal incubation time for the phagocytosis assay. Validate the assay by including positive and negative controls to confirm the reliability of the results. Perform the experiment in biological replicates and calculate the mean value from at least three replicates to ensure accuracy and reliability of the data.

Problem 5

No phagocytosis is observed.

Potential solutions

Coating target cells with opsonizing antibodies or Fc receptor-engaging antibodies can induce basal phagocytosis if experimental conditions allow. It is crucial to use healthy and optimally differentiated macrophages, as poor health or viability can negatively impact phagocytosis. Ensure that the incubation time is sufficient to allow for adequate phagocytic events; adjust the duration as needed to facilitate proper interaction between macrophages and target cells. Additionally, maintain sterile techniques and verify the purity of cell cultures to prevent contamination by other cell types or microorganisms, which can interfere with the phagocytosis process.

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Alok K. Mishra (alok.mishra@umassmed.edu).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Alok K. Mishra (alok.mishra@umassmed.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate any unique datasets or code.

Acknowledgments

This work is dedicated to the memory of Professor Michael R. Green, whose guidance and support were instrumental in its completion. We thank Dr. Michelle Kelliher for providing the necessary resources and support to complete this work. We acknowledge the financial support from the University of Massachusetts Chan Medical School. The graphical abstract was created with BioRender.com.

Author contributions

Conceptualization, A.K.M., S.K.M., and M.R.G.; methodology, A.K.M., S.B., R.P.T., and S.K.M.; formal analysis, A.K.M. and S.K.M.; visualization, A.K.M. and S.K.M.; supervision, M.R.G.; writing – original draft, A.K.M. and S.K.M. All authors read and approved the final manuscript.

Declaration of interests

A.K.M., S.K.M., and M.R.G. are listed as inventors on a patent application filed by the University of Massachusetts Chan Medical School on targeting GPI pathway proteins to treat ovarian cancer.