Protocol to isolate, expand, and activate human type 2 innate lymphoid cells to study immune checkpoint function

Summary

Type 2 innate lymphoid cells (ILC2s) are a subset of innate lymphoid cells involved in allergic diseases, tissue repair, and tumor immunity. Here, we present a protocol to study the function of immune checkpoint receptors on ILC2s in vitro using plate-bound recombinant ligands. We describe steps for the enrichment, expansion, and purification of human ILC2s from healthy donors’ peripheral blood.

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

Graphical abstract

Highlights

• •Steps to enrich ILC2s from HDs PB and subsequently purify them by negative selection
• •Protocol avoids ILC2 flow sorting and the usage of an irradiated allogeneic feeder
• •Steps to study immune checkpoint receptor function on ILC2s using plate-bound ligands

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

Type 2 innate lymphoid cells (ILC2s) are a subset of innate lymphoid cells involved in allergic diseases, tissue repair, and tumor immunity. Here, we present a protocol to study the function of immune checkpoint receptors on ILC2s in vitro using plate-bound recombinant ligands. We describe steps for the enrichment, expansion, and purification of human ILC2s from healthy donors’ peripheral blood.

Before you begin

The percentage of ILC2s among peripheral blood mononuclear cells (PBMCs) is very low, making it very difficult to work with ILC2s in the human setting. The protocol below describes the steps to obtain primary ILC2 cultures to study ILC2 function in vitro. The first step of isolation consists on enriching ILC2s from healthy donors’ (HDs) PB. Culturing ILC2 with “autologous feeder cells” makes the addition of an irradiated allogenic feeder unnecessary. Then we expand ILC2 with cytokines and further purify them by negative selection. We do not stain surface molecules on ILC2s, leaving them completely untouched and fully preserving their functionality. In this protocol, primary ILC2 cultures are generated to study the inhibitory effect of PD-1 and CTLA-4 stimulation on IL-2-induced cytokine release. However, it can also be used to study the effect of other surface receptors. In addition, upon plate-bound recombinant ligand stimulation, modifications in ILC2 phenotype and gene expression can be investigated, respectively, by flow cytometry and real-time PCR or bulk RNA-sequencing.

Innovation

The innovation of our protocol to generate primary ILC2 cultures is that, differently from other published methods, it allows to avoid both (i) the usage of irradiated allogenic feeder cells for ILC2 expansion and (ii) flow-sorting for ILC2 purification, leaving surface antigens totally untouched.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
lineage cocktail 1-FITC CD3 (clone SK7), CD14 (clone MφP9), CD19 (clone SJ25C1), CD20 (clone L27), CD56 (clone NCAM16.2), CD16 (clone 3G8) (diluted 1:10)	BD Biosciences	Cat# 340546; RRID: AB_400053
CD294 (CRTH2)-PE (clone: BM16) (diluted 1:50)	Miltenyi	Cat#130-114-126; RRID: AB_2751204
KLRG1-APC (clone: REA261) (diluted 1:50)	Miltenyi	Cat#130-117-703; RRID: AB_2652574
CD161-PE (clone: 191B8) (diluted 1:50)	Miltenyi	Cat#130-092-677; RRID: AB_2733772
CD7-PE-CF594 (clone: M-T701) (diluted 1:50)	BD Biosciences	Cat#562541; RRID: AB_2737642
GATA3-PE (clone: TWAJ) (diluted 1:20)	eBiosciences	Cat#12-9966-42; RRID: AB_1963600
CD279 (PD-1)-PE (clone: PD1.3.1.3) (diluted 1:50)	Miltenyi	Cat#130-117-384; RRID: AB_2727929
CD152 (CTLA-4)-APC (clone: BNI3) (diluted 1:20)	Miltenyi	Cat#130-123-812; RRID: AB_2857639
Biological samples
Peripheral blood samples of healthy donors
Chemicals, peptides, and recombinant proteins
Ficoll-Hypaque	Lympholyte-H, Cederlane	Cat#CL5020
DPBS (Dulbecco’s Phosphate buffered saline)	Euroclone	Cat#ECB4004L
FBS (Fetal Bovine Serum)	Gibco	Cat#26140-079
BSA (Bovine serum albumin)	Sigma-Aldrich	Cat#A8022-100G
penicillin—streptomycin	Euroclone	Cat#ECB3001D
GlutaMAX	Gibco	Cat#35050-061
L-glutamine	Euroclone	Cat#ECB3000D
CTS NK-Xpander basal medium	Gibco	Cat#A5019001
RPMI 1640 Medium	Euroclone	Cat#ECM9106L
Recombinant human IL-2	R&D	Cat#202-IL
Recombinant human IL-7	Miltenyi	Cat#130-095-362
Recombinant human IL-1β	Miltenyi	Cat#130-095-374
EDTA 0.5M pH 8	Invitrogen	Cat#15575020
rhB7-H1/Fc (PDL-1 FC)	R&D	Cat#156-B7
rhPDL-2 FC	R&D	Cat#1224-PL
rhB7-1/Fc (CD80 FC)	R&D	Cat#10133-B1
rhB7-2/Fc (CD86 FC)	R&D	Cat#7625-B2
rhIgG1 Fc	Biotechne	Cat#110-HG
Human serum albumin (HSA)	Sigma-Aldrich	A9731-1G
LIVE/DEAD Fixable Blue Dead Cell Stain Kit (diluted 1:100)	Invitrogen	Cat#L23105; RRID: AB_3717566
Critical commercial assays
ILC2 Isolation Kit human	Miltenyi	Cat#130-114-825
LEGENDplex HU Th cytokine panel (12-plex)	BioLegend	Cat#741028
EasySep Human ILC2 Enrichment Kit	StemCell Technologies	Cat#17972
Software and algorithms
CytExpert v. 2.5.0.77	Beckman Coulter	N/A
Biolegend Legendplex software	Biolegend	N/A
FlowJo, v. 10.8.1	BD Life Sciences	N/A
Other
Flow cytometer Cytoflex LX	Beckman Coulter	Cat#C00445
The Big Easy	Stemcell	Cat#18001
MACS columns and separators	Miltenyi
96 well plates, flat bottom, high binding	Corning	Cat#3361
96 well Cell Culture Plate round-bottom	Corning Incorporate	Cat#3799
Culture Plate 24 well	Biofil	Cat#TCP011024
Conical Centrifuge Tube 15 mL	FALCON	Cat#352096
Conical Centrifuge Tube 50 mL	FALCON	Cat#352070

Materials and equipment

Complete CTS NK-Xpander basal medium

Reagent	Final concentration	Amount
CTS NK-Xpander basal medium	N/A	430 mL
Supplement	1:50	10 mL
FBS	10%	50 mL
GlutaMAX	1:100	5 mL
penicillin—streptomycin	1:100	5 mL
Total	N/A	500 mL

Store at −4°C for up to 1 month.

Complete RPMI 1640 Medium

Reagent	Final concentration	Amount
RPMI 1640 Medium	N/A	440 mL
FBS	10%	50 mL
Glutamine	1:100	5 mL
penicillin—streptomycin	1:100	5 mL
Total	N/A	500 mL

Store at −4°C for up to 1 month.

EasySep Buffer

Reagent	Final concentration	Amount
PBS	N/A	489 mL
FBS	2%	10 mL
EDTA	1 mM	1 mL
Total	N/A	500 mL

Store at −4°C for up to 3 months.

Miltenyi Buffer

Reagent	Final concentration	Amount
PBS	N/A	497.5 mL
FBS	0.5%	2.5 mL
EDTA	2 mM	2 mL
Total	N/A	500 mL

Store at −4°C for up to 3 months.

Note: Keep buffer cold (2°C–8°C).

Staining buffer

Reagent	Final concentration	Amount
PBS	N/A	497.5 mL
FBS	0.5%	2.5 mL
EDTA	2 mM	2 mL
Total	N/A	500 mL

Store at −4°C for up to 3 months.

PBS and Ficoll should be prewarmed at 37°C.

Step-by-step method details

Enrichment of human ILC2s

Timing: 2 h

This step describes human ILC2 enrichment from PBMCs isolated from HDs buffy coats, using EasySep Human ILC2 Enrichment Kit.

• 1.Transfer 40 ml of blood into 50 mL tubes.
• 2.Dilute blood with prewarmed PBS 1:3.
• 3.Prepare 4 50 mL tubes, pour 15 mL of prewarmed Ficoll in each tube and layer 30 mL of diluted blood on top of Ficoll. The ratio between Ficoll and blood is 1:2.

CRITICAL: Prewarm PBS and Ficoll at 37°C. To avoid mixing of Ficoll and blood, gently add blood to the Ficoll at a 45-degree angle and maintain a constant speed.

• 4.Centrifuge at 1200 g for 20 min at 21°C with acceleration and deceleration rates set to 9 and 3, respectively.

Note: After Ficoll density gradient centrifugation, the blood is stratified into different phases. Cells with a lower density (PBMC) form a ring between an upper plasma phase and a bottom phase with a higher density. Red blood cells (RBCs) with a high density migrate through the density gradient to the bottom (Figure 1).

• 5.Aspirate and discard the upper plasma phase above the ring of PBMC.
• 6.Collect the PBMC ring to a 50 mL tube.
• 7.Wash the PBMC with 30 mL of PBS by centrifugation at 650 g for 5 min.
• 8.Collect the PBMC to a 14 mL round-bottom tube and resuspend 1 × 10⁸ cells/mL in EasySep Buffer.

Note: The minimum volume per 14 mL round-bottom tube for cell enrichment is 1 mL and the maximum is 5 mL. Collect the PBMC to 50 mL tube if the cells are more than 5 × 10⁸. The minimum volume per 50 mL tube for cell enrichment is 5 mL and the maximum is 25 mL. We discourage starting from less than 1 × 10⁸ PBMC because the ILC2 yield may be too low. However, collect the PBMC to 5 mL round- bottom tube if the cells are less than 1 × 10⁸. The minimum volume per 5 mL round-bottom tube for cell enrichment is 0.1 mL and the maximum is 2.5 mL.

• 9.Add Enrichment Cocktail from EasySep Human ILC2 Enrichment Kit to cells: 25 μL per 1 mL of sample and incubate for 10 min at 21°C.
• 10.Add Magnetic Particles from EasySep Human ILC2 Enrichment Kit to cells: 25 μL per 1 mL of sample and incubate for 1 min at 21°C.

Note: Vortex the Magnetic Particles for 1 min before use.

CRITICAL: EasySep Human ILC2 Enrichment Kit is an immunomagnetic negative selection of untouched human ILC2. The protocol provided by STEMCELL Technologies recommends to use 100 μL/mL of Enrichment Cocktail and Magnetic Particles, to obtain ready-to-use pure ILC2s. For our purpose, we suggest to use lower concentrations of both reagents (25 μL/mL), to obtain an enriched, rather than pure, cell suspension. Lower purity allows better ILC2 culture and proliferation conditions thanks to the stimulation by the remaining immune cells which serve as “feeder”.

• 11.Add EasySep Buffer to the sample and gently mix.

Note: Top up to 5 mL for samples less than 2 mL and top up to 10 mL for samples greater than 2 mL.

• 12.Place the 14 mL tube without lid into “The Big Easy” magnet and incubate for 5 min at 21°C to allow labelled non-ILC2s to adhere to the walls of the tube.

Note: An appropriate magnet is chosen according to the number of total cells. Use “The Big Easy magnet” for 14 mL round-bottom tube, use “Easy 50 magnet” for 50 mL tube or “EasySep magnet” for 5 mL round-bottom tube.

• 13.Transfer the medium containing enriched ILC2s to a new 14 mL round-bottom tube by inverting the magnet and tube in a single fluid movement.
• 14.Put the new tube without lid in the magnet and incubate for 5 min at 21°C to allow labelled non-ILC2s to adhere to the walls of the tube for the second time. This step is needed to further enrich the ILC2s.
• 15.Transfer the medium containing enriched ILC2s in a new 14 mL round-bottom tube by inverting the magnet and tube in a single fluid movement.
• 16.Add 10 mL of PBS and centrifugate at 650 g for 5 min at 21°C to wash ILC2.
• 17.Seed the enriched ILC2s in a 96-well round-bottom plate at a density of 2 × 10⁵ cells/200 μL/well in CTS NK-Xpander basal medium with the cytokines IL-2, IL-7, and IL-1β (50 ng/ml each) in the incubator at 37°C and 8% CO₂.

Note: At this stage, the range of total enriched ILC2s number typically falls between 0.1 to 2 × 10⁶, starting from 3.5/4.5 × 10⁸ PBMCs isolated from 40 mL HD buffy coat. Results may vary among donors.

Figure 1.

Ficoll gradient centrifugation

Separation of blood components using Ficoll gradient centrifugation. Created in Biorender.

Expansion of human ILC2s following enrichment

Timing: 10 days + 1 h

This step describes the expansion of human ILC2s and the subsequent cytometer purity assessment, prior to the next purification step.

• 18.Check the cells and their confluence every 1–2 days.

Note: Refresh medium with cytokines every 3 days by replacing half of the media; split cells in more wells to keep their concentration between 3 and 4 × 10⁶ cells/mL.

• 19.
  At day 10, harvest a representative cell aliquot to check the purity of the cell population.

  • a.
    Transfer 30 μL of cells into a FACS tube.
  • b.
    Wash the cells with 1 mL of cold PBS by centrifugation at 650 g for 5 min at 21°C and discard the supernatant.
  • c.
    Prepare an antibody mix with the LIVE/DEAD Fixable Blue Dead Cell Stain Kit (diluted 1:100), anti-lineage cocktail 1 -FITC (diluted 1:10) and anti-CRTH2-PE (diluted 1:50) antibody in staining buffer (consider 20 μL per sample, always prepare at least 10% excess to account for pipetting errors).
  • d.
    Resuspend the cells in 20μL of antibody mix prepared in c and incubate them for 20 min in the dark at 4°C.
  • e.
    Wash the cells with 1 mL of cold PBS by centrifugation at 650 g for 5 min and discard the supernatant.
  • f.
    Resuspend stained cells in 200 μL of cold PBS.
  • g.
    Acquire the cells on a cytometer and analyse flow data using the gating strategy as shown in Figure 2.

Figure 2.

Gating strategy to identify ILC2

ILC2 are identified within the lymphocyte gate as LIN (CD3, CD14, CD19, CD20, CD56, CD16)^- and CRTH2⁺. In (A) and (B) ILC2 pre- and post-separation from HDs buffy coats by EasySep Human ILC2 Enrichment Kit StemCell are shown. In C and D ILC2 pre- and post-separation by Human ILC2 Isolation Kit Miltenyi are shown. In (A), (C) and (D) cells are firstly gated to exclude dead cells. Live cells are gated as negative for LIVE/DEAD Fixable Blue Dead Cell Stain Kit.

Purification of ILC2s by depletion of lineage-positive cells

Timing: 1:30 h

This step describes the procedure of purification of ILC2 by negative selection using the human ILC2 Isolation Kit Miltenyi, labelling the lineage-positive cells with a cocktail of biotin-conjugated antibodies and anti-biotin monoclonal antibodies conjugated to micro beads. Biotin-conjugated antibodies included in the cocktail recognize antigens that are not expressed by ILC2: CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a.

• 20.Harvest enriched ILC2 in sterile conditions in a conical 15 mL tube.
• 21.Wash cells with 10 mL of PBS by centrifugation at 300 g for 10 min at 4°C.
• 22.Resuspend the cells in a final volume of 40 μL of cold Miltenyi buffer (4°C) and 10 μL of biotin-antibody cocktail.

Note: The indicated volumes are for up to 1 × 10⁷ cells. For higher cell numbers, scale up volumes accordingly.

• 23.Mix and incubate for 10 min at 4°C.
• 24.Wash the cells with 2 mL of cold Miltenyi buffer (4°C) by centrifugation at 300 g for 10 min at 4°C.
• 25.Discard the supernatant.
• 26.Resuspend the cells in a final volume of 80 μL of cold buffer Miltenyi and 20 μL of anti-biotin micro beads.

Note: The indicated volumes are for up to 1 × 10⁷ cells. For higher cell numbers, scale up volumes accordingly.

• 27.Mix and incubate for 15 min at 4°C.
• 28.Wash the cells with 2 mL of cold buffer by centrifugation at 300 g for 10 min.
• 29.Discard the supernatant.
• 30.Resuspend cells in 500 μL cold Miltenyi buffer.

Note: The indicated volume is for up to 1 × 10⁸ cells. For higher cell numbers, scale up volume accordingly.

• 31.Place a MS MACS column in the magnetic field of the MACS Separator with a 30 μm filter on top.

CRITICAL: According to the protocol provided by Miltenyi, an appropriate MACS Column and MACS Separator are chosen depending on the number of total labelled cells. The volume of Miltenyi buffer to use for washing depends on the kind of column chosen. We recommend to use MS MACS columns because at this step the starting number of cells is between 3–10 × 10⁶.

• 32.Prepare the filter and the column by rinsing with 500 μL cold Miltenyi buffer.
• 33.Apply the cell suspension onto the column through the filter.
• 34.Wash the column 3 times with 500 μL cold Miltenyi buffer.
• 35.Collect the effluent, containing the unlabelled ILC2.

Note: If ILC2 purity checked as in 18 is lower than 80% repeat the magnetic separation.

• 36.Wash the effluent with PBS by centrifugation at 650 g for 5 min at R.T.
• 37.Culture ILC2s in a 96-well round-bottom plate at a density of 2 × 10⁵ cells/200 μL/well in CTS NK-Xpander basal medium with the cytokines IL-2, IL-7, and IL-1β (50 ng/ml each) in the incubator at 37°C and 8% CO₂ for an additional 4 days.

Note: After purification, the total ILC2 number typically falls between 0.2 to 2 × 10⁶. After 4 days, it typically falls between 1 to 5 × 10⁶. Results may vary among donors.

ILC2 starvation

Timing: 30 min + 72 h

To study the function of immune checkpoint receptors, cells are starved for 72 h before being seeded onto plates coated with recombinant ligands.

• 38.Harvest ILC2 in sterile conditions.
• 39.Wash cells with PBS by centrifugation at 650 g for 5 min and discard the supernatant.
• 40.Place ILC2s in 24-well plates at a density of 1 × 10⁶ cells/1 mL/well in complete RPMI 1640 Medium for 72 h.

CRITICAL: Cell starvation from cytokines is important to decrease ILC2 basal activation status and better appreciate the effect of positive and negative stimulation.

Plate coating

Timing: 30 min + 18 h

Each receptor is individually triggered using recombinant Fc ligand–chimeric proteins to assess the inhibitory effects of PD-1 and CTLA-4. Plate is coated with the recombinant human ligands 18 h before cell seeding.

• 41.Prepare the recombinant human ligands PD-L1, PD-L2, CD80, CD86, and IgG1 (negative control) at 10 μg/ml each in cold PBS.

Note: In addition to IgG1, human serum albumin (HSA) can be used as a negative control of a generic protein.

• 42.Add 50 μl/well of each coating solution in a 96-well flat-bottom high-binding plate.
• 43.Seal with Parafilm the plate and incubate at 4°C overnight to allow coating.

Cell seeding and stimulation

Timing: 1 h + 24 h

After starvation, ILC2s are seeded to be stimulated by plate-bound recombinant ligands.

• 44.Aspirate the coating solution from wells.
• 45.Wash each well twice with 200 μl cold PBS.
• 46.Add 50 μl of warm complete RPMI 1640 Medium and incubate at 37°C for 30 min.

Note: Pre-incubating each well with FBS-containing medium for 30 min beforehand is important to block nonspecific binding.

• 47.Harvest ILC2 obtained in point 40 in sterile conditions and wash the cells with PBS by centrifugation at 650 g for 5 min at 21°C.
• 48.Resuspend the cells at a density of 5 × 10⁴ cells/150 μL/well in Complete RPMI 1640 Medium with IL-2 (10 ng/ml), seed in the pre-coated plate, and incubate for 24 h at 37°C.
• 49.Transfer the volume of each well in an Eppendorf tube and centrifuge at 650 g for 5 min.
• 50.Collect cells and supernatants for subsequent applications.
• 51.Analyse the cytokine in the supernatants with LEGENDplex HU Th cytokine panel (12-plex) following the manufacturer’s protocols.

Note: Analyse supernatants immediately or stored frozen for later analysis. Freezing does not significantly alter cytokine concentrations.

• 52.Analyse ILC2 phenotype by flow cytometry and cell gene expression by RNA extraction, RT-PCR and real-time PCR or bulk RNA-sequencing.

Note: Suitable markers that can be analysed include activating receptors such as ICOS and inhibiting receptors such as PD-1, CTLA-4 and other immune checkpoints (by flow cytometer) and the genes IL4, IL5, IL13 (by Real Time PCR). Additional markers and genes can be analysed depending on the purpose of the experiments.

Expected outcomes

After ILC2 enrichment, expansion and further isolation, we obtained a purity of around 95%. ILC2s were characterized as LIN(CD3, CD14, CD19, CD20, CD56, CD16)^- and CRTH2⁺ cells (Figure 3). In addition, we observed that ILC2 cultures that we have generated in vitro expressed the lymphocyte marker CD7, and CD161 and KLRG1, both markers normally expressed on ILC2 (Figure 3). Moreover, this population expressed the transcription factor GATA3, important for ILC2 development and functions. We also observed that activated primary ILC2 express the immune checkpoints PD-1 and CTLA-4 (Figure 4). The role of these checkpoints on ILC2 is not very clear yet. We used this protocol to stimulate ILC2 with IL2 for 24 h in the presence of plate-bound PDL1, PDL2, CD80, and CD86 recombinant Fc chimeric proteins, and IgG-Fc as a control, to confirm that they have an inhibitory role. After stimulation, we analysed in the supernatants the concentrations of IL-5 and IL-13 released by ILC2 in response to IL-2 stimulation. We found that, as expected, cytokine release decreased in the presence of each ligand compared to Ig control (Figure 5). Other recombinant proteins can also be used as plate-bound ligands.

Figure 3.

Characterization of primary ILC2 cultures

(A) Expression of CRTH2, KLRG1, CD7, CD161 and LIN (CD3, CD14, CD19, CD20, CD56, CD16) on cultured ILC2 and (B) expression of GATA3 in cultured ILC2. In A and B, the grey histograms show FMO as a negative control, and the red histograms show ILC2. FACS plots are shown for one representative HD.

Figure 4.

Expression of immune checkpoints on primary ILC2 cultures

PD-1 and CTLA-4 expression on cultured ILC2. The grey histograms show FMO as a negative control, and the blue histograms show ILC2. FACS plots are shown for one representative HD.

Figure 5.

PD-1 and CTLA-4 ligands inhibit ILC2 cytokine release

Concentrations of IL-5 and IL-13 released in supernatants by ILC2, stimulated with IL-2 for 24 h in the presence of plate-bound PD-L1, PD-L2, CD80 and CD86 recombinant Fc chimeric proteins, and IgG-Fc as a control. The histograms show one representative experiment.

Limitations

The main risk associated with the generation of ILC2 primary cultures is the low ILC2 yield to be able to perform experiments. Moreover, there is a variability in ILC2 abundance and expansion depending on the donor. Therefore, it is important to isolate ILC2 from different donors in parallel to be sure to isolate a sufficient number of ILC2 from at least one of them and generate ILC2 cultures frequently to perform experiments.

Troubleshooting

Problem 1

Low cell yield (step 37).

Potential solution

If the ILC2 yield is low, try to increase the cell concentration during culture. High cell density enhances interaction between cells, and ILC2s require cell-cell contact to grow. Moreover, try to refresh the medium and cytokines more frequently.

Problem 2

ILC2 purity lower than 80% (step 35).

Potential solution

After ILC2 purification by magnetic beads, the expected ILC2 purity is 95%. This percentage may vary between different donors. In case of low ILC2 purity (lower than 80%), magnetic separation can be repeated.

Problem 3

No inhibition or activation of ILC2s is observed in the plate-bound ligand assay (step 41).

Potential solution

For each protein, different concentrations for plate coating should be tested in a preliminary experiment to find the optimal conditions of ILC2 activation/inhibition.

Problem 4

It is needed to dilute the samples to make the cytokines concentration fall within the detectable range of the standard curve (step 51).

Potential solution

The concentrations of analytes in ILC2 supernatants can vary greatly between different donors and from the activation state of the cell. A single dilution of the supernatant may not be sufficient to ensure that the sample concentration falls within the standard curves’ detectable and linear ranges for different analytes, especially when they are measured simultaneously (as with the LEGENDplex HU Th cytokine panel). Therefore, a preliminary experiment may be needed to determine the appropriate dilution factors.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Linda Quatrini (linda.quatrini@opbg.net).

Technical contact

Technical questions about this protocol should be directed to the technical contact, Cecilia Ciancaglini (cecilia.ciancaglini@opbg.net).

Materials availability

This protocol did not generate new materials.

Data and code availability

The study did not generate/analyze datasets or codes.

Acknowledgments

This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC): AIRC MFAG ID 27022 (L.Q.), AIRC IG Grant ID 27065 (P.V.), and AIRC 5X1000 2018 ID 21147 (L.M.). This work was funded by the European Union—Next Generation EU—NRRP M6C2—Investment 2.1 Enhancement and strengthening of biomedical research in the NHS by the Italian Ministry of Health (PNRR-MCNT2-2023-12378122) (L.Q.). This work was supported by the Italian Ministry of Health with “Current Research Funds.” S.S. is the recipient of a fellowship awarded by Fondazione Veronesi.

Author contributions

C.C. wrote the paper, and L.Q. reviewed and edited it. All authors discussed the results and commented on the manuscript.

Declaration of interests

The authors declare no competing interests.