Protocol for modeling innate immune training by repeated alum exposure in mice (adjuvant conditioning)

Summary

We present a protocol to model the effects of innate immune training and reprogramming through repeated alum exposure in mice, enabling the evaluation of immunoregulatory cell populations in both steady-state and inflammatory conditions. We describe steps for integrating adoptive cell transfer, in vivo antigen-specific immunization, and transplant models. We also detail procedures for in vitro stimulation of human peripheral blood mononuclear cells (PBMCs) to explore translational relevance.

For complete details on the use and execution of this protocol, please refer to Boccia et al.¹ and Boccia et al.²

Graphical abstract

Highlights

• •Steps for adjuvant conditioning/innate immune training via repeated adjuvant exposure
• •Instructions for myeloid cell isolation adjuvant conditioning
• •Testing of adjuvant conditioning efficacy through alloislet transplant model
• •Guidance on in vitro and in vivo T cell suppression assays

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

We present a protocol to model the effects of innate immune training and reprogramming through repeated alum exposure in mice, enabling the evaluation of immunoregulatory cell populations in both steady-state and inflammatory conditions. We describe steps for integrating adoptive cell transfer, in vivo antigen-specific immunization, and transplant models. We also detail procedures for in vitro stimulation of human peripheral blood mononuclear cells (PBMCs) to explore translational relevance.

Before you begin

This protocol describes the detailed steps for performing adjuvant conditioning (AC) in mice, followed by downstream immunological assays including pancreatic islet transplantation, adoptive cell transfer, flow cytometry, cytokine quantification, delayed-type cutaneous hypersensitivity (DTH), and in vitro stimulation of human peripheral blood mononuclear cells (PBMCs). Experiments are conducted in mice of various genetic backgrounds and supplemented with human samples to assess translational relevance. For complete details on the use and execution of this set of protocols, please refer to Boccia et al.¹

Innovation

This protocol provides a streamlined and translationally relevant approach to study alum-induced immunomodulation, integrating in vivo murine conditioning with in vitro functional analysis of myeloid-derived suppressor cells (MDSCs) and adaptive immune responses. Unlike conventional approaches that examine either animal models or human immune cells in isolation, this workflow unifies alum preconditioning, MDSC purification, T cell suppression assays, and antibody quantification into a single, reproducible framework. Key innovations include an optimized alum dosing regimen that reliably establishes systemic immunosuppression, a combined mechanical and chemical method for isolating viable immune cells for downstream assays and standardized co-culture setups to quantitatively evaluate MDSC-mediated inhibition of CD4⁺ T cell proliferation. The protocol also incorporates translational modeling using human PBMCs to assess alum-driven modulation of inflammatory cytokines, bridging mechanistic findings in mice with human immune responses. By merging established techniques into a coherent, adaptable workflow, this protocol enables comprehensive evaluation of innate and adaptive immunoregulation, offering a versatile platform for preclinical studies of adjuvant effects, immune tolerance, and regulatory myeloid cell function. This represents an advance over existing methods by providing a single, integrated approach that is both reproducible and broadly applicable to experimental and translational immunology.

Institutional approvals

All mouse experimental protocols were approved by the Institutional Animal Care and Use Committees of Boston Children’s Hospital under protocol number 20-01-4117/00001847. Human PBMC samples (buffy coat donations) were collected following written informed consent from each donor. The protocols for sample collection were approved in advance by the local medical ethics committee (IRB-P00038916), in accordance with the Declaration of Helsinki.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
CD11b PE (1:200)	Invitrogen, clone M1/70	12-0112-82; RRID:AB_2734869
Ly6C PeCy7 (1:100)	BioLegend, clone HK1.4	128016; RRID:AB_1732076
Ly6G PercP-Cy5 (1:100)	BioLegend, clone 1A8	127616; RRID:AB_1877271
CD3 FITC (1:100)	Invitrogen, clone 17A2	11-0032-82; RRID:AB_2572431
CD4 PeCy7 (1:100)	Invitrogen, clone GK1.5	25-0041-82; RRID:AB_469576
TCR Va2 PE (1:100)	BioLegend, clone B20.1	127806; RRID:AB_1134184
TCR Va2 PercP-Cy5 (1:100)	BioLegend, clone B20.1	127808; RRID:AB_1186118
HRP Goat anti-mouse IgG (1:10000)	Sigma-Aldrich	A4416; RRID_AB_258167
Purified anti-IDO Antibody	BioLegend, clone 2E2/IDO1	122402; RRID:AB_2280140
Purified anti-GAPDH Antibody	BioLegend, clone W17079A	607901; AB_2734502
Biological samples
Human PBMCs	Leukoreduction System Cones from Boston Children’s Hospital blood bank
Chemicals, peptides, and recombinant proteins
Alum Adjuvant	G-Biosciences	786–1215
Lysis buffer ACK	Gibco	A1049201
STZ	Sigma	S0130
Saline	Gibco	14190250
LPS	Sigma	L4391
OVAp	Invivogen	vac-pova
OVA-V	Sigma	V1701
RPMI-1640 Medium	Corning	10-040-CV
Penicillin-Streptomycin (10000 U/mL)	Gibco	15140122
Fetal Bovine Serum	Gibco	26140079
Sodium Pyruvate (100 mM)	Gibco	11360070
2ME (2-Mercaptoethanol)	Gibco	21985023
Lymphoprep density gradient medium centrifugation	STEMCELL Technologies	07801
EDTA	Sigma-Aldrich	E9884
PBS 10x	Corning	46-013-CM
Tween-20	Sigma-Aldrich	P1379
Carbonate-Bicarbonate Buffer with Azide BioUltra, tablet	Sigma-Aldrich	08058
OPD (o-Phenylenediamine Dihydrochloride)	Sigma-Aldrich	P8287
TBS 10X, pH 7.4, DNase/RNase and protease free	Corning	46-012-CM
BSA (Bovine Serum Albumin)	Sigma-Aldrich	A2153
Sulfuric Acid, 99.999%	Sigma-Aldrich	339741
Critical commercial assays
Fixation/Permeabilization Kit	BD Biosciences	554714
Mouse MDSC Enrichment Kit	StemCell Technologies	19762
Mouse Naïve CD4+ T Cell Isolation Kit	StemCell Technologies	19765
Fixable Viability Dye (FVD) eFluor™ 780	Invitrogen	65-0865-14
Zombie Violet™ Fixable Viability Kit (1:1000)	BioLegend	423113
CellTrace™ Violet Cell Proliferation Kit, for flow cytometry	Invitrogen	C34557
Accu-Chek Guide Meter	Accu-check	07562462001
Accu-Chek Guide Test Strips (50 count)	Accu-check	07453744119
Experimental models: Organisms/strains
C57BL/6J (H-2b) mice (male or female 8-10 week-old)	Jackson Laboratory	#000664
Balb/C (H-2d) mice (male or female 8-10 week-old)	Jackson Laboratory	#000651
OT-II (B6.Cg-Tg(TcraTcrb)425Cbn/J) (male or female 8-10 week-old)	Jackson Laboratory	#004194
Software and algorithms
LSRFortessa HTS with FACS Diva	BD Biosciences	v8.0.2
FlowJo	BD Biosciences	v10
Graphpad Prism	Graphpad	v10
Affinity Designer	Affinity Designer	V1.10.8
Microsoft Word	Microsoft	2016
Microsoft Excel	Microsoft	2016
Fluostar Omega	BMG Labtech
Other
Countess Automated Cell Counter	Thermo Fisher Scientific	AMQAX1000
Countess Cell Counting Chamber Slides	Thermo Fisher Scientific	C10228
Class II Biosafety Cabinet	Thermo Fisher Scientific	1386
Refrigerated Benchtop Centrifuge	Eppendorf	5425 R
−20°C Freezer	Thermo Fisher Scientific	TSX2320FA
−80°C Ultra-Low Freezer	Thermo Fisher Scientific	TSX70086A
Water bath, 37°C	VWR	89032–208
Dry block heater	Thermo Fisher Scientific	88870003
Benchtop Vortex Mixer	VWR	10153–838
pH Meter	Mettler Toledo	FiveEasy F20
Vacuum Aspirator System	VWR	89130–938
CO2 euthanasia chamber	Plexx	TT-8200
Dissection board	Fisher Scientific	36–119
Forceps, 45° angled serrated, 16 cm	FST	11080–02
Forceps, straight serrated, 13 cm	FST	11000–13
Forceps, fine straight serrated, 13 cm	FST	11008–13
Scissors, straight blunt, 11 cm	FST	14074–11
Scissors, straight sharp, 8.5 cm	FST	14084–08
Magnetic stirrer	IKA	3622000
Rainin Single-Channel Pipette, 0.1–10 μL (LTS)	Rainin	L-10XLS+
Rainin Single-Channel Pipette, 10–100 μL (LTS)	Rainin	L-100XLS+
Rainin Single-Channel Pipette, 20–200 μL (LTS)	Rainin	L-200XLS+
Rainin Single-Channel Pipette, 100–1000 μL (LTS)	Rainin	L-1000XLS+
Rainin Multichannel Pipette, 20–200 μL (LTS, 8-channel)	Rainin	L8-200XLS+
Rainin Multichannel Pipette, 10–100 μL (LTS, 8-channel)	Rainin	L8-100XLS+
Mouse lab animal cages (Eurostandard type II L)	Tecniplast	1284L
Roker 2D Digital rocking shaker	IKA-Werke GmbH	4003000
1.5 mL Eppendorf tubes	Eppendorf	0030121023
15 mL Falcon conical polypropylene tubes	Falcon	352096
5 mL polypropylene tubes	Corning	352002
50 mL Falcon conical polypropylene tubes	Falcon	352070
12-well plates, non-treated	Falcon	351143
60-mm Petri dishes	Falcon	353004
96-well plates, flat-bottom, non-treated	Fisher Scientific	267578
96-well plates, round-bottom, non-treated	Corning	3799
RIPA buffer + protease/phosphatase inhibitors	Thermo Fisher Scientific	Cat# 89900 + Cat# 78440 (PI) + Cat# 78420 (PPI)
Pierce™ BCA Protein Assay Kit	Thermo Fisher Scientific	Cat# 23225
SDS-PAGE reagents and apparatus	Bio-Rad	Mini-PROTEAN® TGX Gels, Cat# 4561094; Gel Electrophoresis Cell, Cat# 1658004
PVDF membrane (0.45 μm)	MilliporeSigma (Merck)	Cat# IPVH00010
ECL substrate	Thermo Fisher Scientific	Cat# 34095
ChemiDoc imaging system	Bio-Rad	Cat# 12003154

Materials and equipment

Complete RPMI

Component	Final concentration	Stock concentration	Volume per 500 mL	Volume per 1 L
RPMI-1640 (base medium)	—	—	500 mL	1 L
Fetal Bovine Serum (FBS)	10% (v/v)	100%	50 mL	100 mL
Penicillin–Streptomycin	1% (v/v)	100×	5 mL	10 mL
Sodium Pyruvate	1 mM	100 mM	5 mL	10 mL
2-Mercaptoethanol (β-ME)	50 μM	50 mM	0.5 mL	1 mL
HEPES buffer	10 mM	1 M	5 mL	10 mL

[Note on storage conditions: Store complete media at 4°C for up to one month].

OPD Working Solution Recipe

Component	Final concentration	Stock concentration	Volume per 10 mL	Volume per 50 mL
Citrate–phosphate buffer	0.05 M, pH 5.0	—	10 mL	50 mL
OPD tablet	1 tablet per 10 mL (10mg/mL)	—	1 tablet	5 tablets
H2O2	0.03% (v/v)	30% stock	30 μL	150 μL

[Note on storage conditions: Store citrate-phosphate buffer at 25°C and H₂O₂ at 4°C for up to six months. Unused OPD working solution should be discarded after use].

Step-by-step method details

Animal preparation and adjuvant administration

Timing: 5 days (3 injections every other day)

This step aims to establish a systemic immunosuppressive state in mice through repeated intraperitoneal administration of alum. Alum, an aluminum-based adjuvant, induces a mild chronic inflammatory response that leads to immune deviation and tolerance upon repeated exposure. This preconditioning mimics an immunosuppressed environment that facilitates subsequent experimental manipulations by dampening adaptive immune activation and skewing innate immune responses.

• 1.Randomization: Randomly assign mice to treatment (alum), control (saline) groups with at least 5 mice per group.
• 2.Housing: Place 8-10 week-old male or female C57BL/6 mice under specific pathogen-free conditions with ad libitum access to food and water.
• 3.
  Injection: Administer intraperitoneal injections as follows:

  • a.
    AC: 8 mg alum in 200 μL volume.
  • b.
    Control: 200 μL sterile saline.
  • c.
    Schedule: Day 0, Day 2, Day 4 (every other day for 3 injections) (Figure 1A).
• 4.Monitoring: Observe mice daily for signs of distress: brief hunching, stillness, reduced exploratory behavior, temporary abdominal sensitivity.
• 5.Documentation: Record injection time, volume, and any adverse events.

CRITICAL: Maintain consistent injection timing across all mice to minimize experimental variability.

Figure 1.

Adjuvant Conditioning Protocol

(A) Experimental design: 8–10-week-old male C57BL/6J mice were injected intraperitoneally (i.p.) with either 8 mg of Alum Imject™ in 200 μL saline or saline alone, three times on alternate days. Spleens were collected on day 5 after the first injection for downstream analysis.

(B) Representative image of spleens from mice injected with either saline or alum on alternate days for a total of three injections.

Preparation of single-cell suspensions from spleens

Timing: Approximately 1 h per 15 mice

This step aims to obtain a viable single-cell suspension from the collected tissue that is suitable for downstream applications such as flow cytometry and in vitro stimulation assays. Mechanical dissociation is used to disrupt the tissue and release immune cells, followed by treatment with ACK buffer to remove residual red blood cells. Proper filtration and washing steps are essential to remove debris and aggregates, ensuring accurate flow cytometric analysis and reliable functional readouts in subsequent assays.

• 6.Euthanize mice using CO₂ euthanasia chamber followed by cervical dislocation as a secondary method.
• 7.Immediately harvest spleens under aseptic conditions and place in cold PBS.
• 8.Mince spleens syringe plunger (1mL of cold PBS per spleen) and press tissue and liquid through a 70 μm cell strainer into a 50 mL tube containing cold PBS.
• 9.Centrifuge at 300 × g for 5 min at 4°C. Discard supernatant carefully.
• 10.Resuspend cell pellet in 3 mL ACK lysis buffer and incubate for 5 min at 25°C to lyse red blood cells.
• 11.Quench lysis by adding 10 mL cold PBS or RPMI medium.
• 12.Centrifuge again at 300 × g for 5 min at 4°C. Discard supernatant.
• 13.Wash cells twice in PBS by resuspension and centrifugation.
• 14.Resuspend cells in PBS for downstream applications (5mL per spleen).
• 15.Count viable cells using trypan blue exclusion.

CRITICAL: Avoid overexposure to ACK buffer (>5 min) to prevent leukocyte damage.

Flow cytometry staining and acquisition

Timing: Approximately 2 days

This step aims to phenotype immune cell populations based on the expression of specific surface and intracellular markers using flow cytometry. Surface staining allows identification of major leukocyte subsets and activation states, while intracellular staining enables the detection of cytokines, transcription factors, or other intracellular proteins of interest. Together, these analyses provide a comprehensive overview of immune cell composition and functional status within the sample.

• 16.Transfer 1 × 10⁶ cells per sample to a 96-well round bottom plate.
• 17.Wash cells with PBS + 3% FBS (FACS Buffer) and centrifuge at 300 × g for 5 min.
• 18.Incubate cells with Fc block (anti-CD16/32) for 15 min on ice.
• 19.Wash cells with PBS + 3% FBS (FACS Buffer) and centrifuge at 300 × g for 5 min.
• 20.Add viability dye (1:1000 in 20uL per well) and incubate for 30 min at 4°C in the dark.
• 21.Wash with 200uL Facs Buffer and centrifuge at 300 × g for 5 min.
• 22.Add surface antibody cocktail in concentration listed in the Key Resource Table and incubate for 30 min at 4°C in the dark.
• 23.Wash with 200uL Facs Buffer and centrifuge at 300 × g for 5 min.
• 24.When staining for surface markers, skip to 11.
• 25.
  When performing an intracellular staining:

  • a.
    Resuspend cells in 100 μL of BD Cytofix/Cytoperm™ solution (1:1 diluted from the stock with distilled water).
  • b.
    Incubate for 20 min at 4°C in the dark.
  • c.
    Add 100 μL 1× BD Perm/Wash buffer (provided in the kit) and centrifuge at 300 × g for 5 min.
• 26.Resuspend cells in Perm/Wash buffer containing fluorochrome-conjugated antibodies against intracellular targets (e.g., cytokines such as IFN-γ, IL-17).
• 27.Incubate for 30–45 min at 4°C in the dark.
• 28.Wash twice with Perm/Wash buffer and centrifuge at 300 × g for 5 min.
• 29.Resuspend the stained cells in 200 μL FACS Buffer and transfer samples to 5mL round bottom tubes.
• 30.Store at 4°C protected from light until acquisition.
• 31.Vortex and acquire samples on a flow cytometer within 24 hours (100,000 events).
• 32.Analyze using FlowJo, employing FMO and negative controls for gating (Figures 2A and 4B).

CRITICAL: Include unstained, single-stain, and FMO to ensure accurate gating.

Figure 2.

Phenotyping and Isolation of Myeloid-Derived Suppressor Cells (MDSCs)

(A) Flow cytometry gating strategy to identify and isolate monocyte-like MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). Starting from single, viable cells, MDSCs were identified as CD11b⁺Ly6C⁺Ly6G^- (M-MDSCs) and CD11b⁺Ly6C⁺Ly6G⁺ (PMN-MDSCs).

(B) Materials used for MDSC isolation from mouse splenocytes, including the EasySep™ Mouse MDSC (CD11b⁺GR1⁺) Isolation Kit, EasySep™ Magnet, and 5 mL round-bottom tubes.

(C) Purity assessment of CD11b⁺GR1⁺ cells before and after isolation using flow cytometry.

Figure 4.

In Vivo Assessment of Adjuvant Conditioning Effects

(A) Experimental timeline: 8–10-week-old male C57BL/6J mice were injected i.p. with either 8 mg of Alum Imject™ or saline every other day (three total injections). Twenty-four hours after the last injection, mice received 5 × 10⁶ OT-II splenocytes intravenously. Another 24 hours later, mice were immunized subcutaneously with 10 μg of OVA adsorbed to 1.5 mg of Alum Imject™ on days 1 and 8 (prime-boost). Mice were euthanized on day 15 for spleen and serum collection.

(B) Flow cytometry gating strategy to identify OVA-specific CD4⁺ T cells following immunization.

(C) Experimental setup for assessing the immunosuppressive effects of adoptively transferred CD11b⁺GR1⁺ cells. Mice received 2 × 10⁶ CD11b⁺GR1⁺ cells isolated from alum-conditioned (AC), or saline-treated WT mice. On the same day, mice were also adoptively transferred with OT-II cells and immunized as described. Immune responses were evaluated on day 15.

MDSC isolation and culture

Timing: 3 h

This step aims to isolate and culture myeloid-derived suppressor cells (MDSCs) for downstream functional studies. Purification is performed to enrich for MDSCs based on characteristic surface markers, minimizing contamination from other myeloid or lymphoid populations. Culturing the purified MDSCs under defined conditions preserves their suppressive phenotype and viability, enabling accurate assessment of their immunomodulatory functions in subsequent in vitro assays.

• 33.
  MDSCs isolation from saline or AC-treated splenocytes (Figure 1B).

  • a.
    Count cells and resuspend up to a maximum of 1.5 × 10⁸ cells per 100 μL of staining buffer 5mL round-bottom tube.
  • b.
    Add to sample:

    • i.
      40 μL FcR blocker per 1 mL of sample.
    • ii.
      50 μL of Isolation Cocktail per 1 mL of sample.
  • c.
    Mix gently and incubate for 10 minutes at 25°C.
  • d.
    Vortex MDSC enrichment particles and add 75 μL to 1 mL of sample.
  • e.
    Mix gently and incubate for 5 minutes at 25°C.
  • f.
    Add staining buffer to bring the total volume up to 2.5 mL. Mix gently.
  • g.
    Place the tube in the EasySep™ magnet without the cap and incubate for 5 minutes.
  • h.
    Without disturbing the magnet or the pellet, carefully pour the enriched MDSC-containing supernatant into a new tube.

Note: The supernatant contains your untouched MDSCs (CD11b⁺Gr-1⁺), ready for downstream applications such as flow cytometry, and suppression assays.

CRITICAL: Leave the magnet and tube inverted for 2–3 s, then return upright. Do not shake or blot off any drops that may remain hanging from the mouth of the tube. Verify cell viability and phenotype before use in functional assays.

• 34.
  MDSCs culture in vitro.

  • a.
    Culture cells at 3 × 10⁵ cells/mL in RPMI-1640 complete medium (RPMI 1640, 1% Penicillin-Streptomycin, 50mM 2-Mercaptoethanol, 1mM Sodium Pyruvate, 10% Fetal Bovine Serum. Keep media at 4°C).
  • b.
    Stimulate with 200 ng/mL LPS for 24h as indicated.
  • c.
    Collect supernatants and cells for downstream assays.

Note: Typical purity is >85% CD11b⁺Gr-1⁺ cells, depending on tissue source, mouse strain and/or treatment (Figure 2C). Yield will vary; spleens typically give approximately 0.1–1 million MDSCs per mouse under inflammatory or tumor-bearing conditions.

T cell suppression assay using MDSCs

Timing: 8 days

This step aims to evaluate the suppressive capacity of MDSCs on CD4⁺ T cell proliferation in vitro. Purified MDSCs are co-cultured with activated CD4⁺ T cells at defined ratios, allowing assessment of their ability to inhibit T cell expansion. Measuring T cell proliferation—typically through dye dilution, and flow cytometric analysis—provides a quantitative readout of MDSC-mediated suppression and reflects their functional activity in modulating adaptive immune responses.

• 35.Prepare a single-cell suspension from male OT-II splenocytes and C57BL/6J total splenocytes as described above.
• 36.
  Naïve CD4 T cell isolation.

  • a.
    Count cells and resuspend at 1×10⁸ total cells per 2.5 mL of PBS per separation. For higher volumes, scale up reagents accordingly.
  • b.
    Place the tube into the CD4 enrichment magnetic magnet and incubate for 2.5 min.
  • c.
    Without disturbing the magnet, carefully pour off the supernatant into a new tube. This supernatant contains the untouched enriched naïve CD4⁺ T cells.
  • d.
    Discard the magnet-bound cells.
  • e.
    Count and check purity/viability (see flow cytometry staining step) (Figure 3B).
• 37.
  CellTrace staining.

  • a.
    Prepare the CellTrace™ Violet Stock Solution.

    • i.
      Reconstitute the lyophilized CellTrace™ Violet dye with 20 μL of DMSO to make a 5 mM stock solution.
    • ii.
      Mix by vortexing for 1 min. Aliquot and store at −20°C protected from light.
  • b.
    Wash freshly isolated naïve CD4⁺ T cells once with PBS (no FBS).
  • c.
    Resuspend cells at 1 × 10⁶ cells/mL in pre-warmed PBS (no FBS).
  • d.
    Dilute CTV stock solution in PBS to achieve a final staining concentration of 5 μM in the cell suspension.
  • e.
    Incubate at 37°C for 20 min in the dark.
  • f.
    Add 5x the staining volume of pre-warmed complete culture medium (e.g., 5 mL for 1 mL of cells).
  • g.
    Incubate for 5 min at 37°C to allow unbound dye to be quenched.
  • h.
    Centrifuge at 300 × g for 5 min.
  • i.
    Wash cells twice with complete medium or PBS + 2% FBS to remove excess dye.
• 38.
  Co-culture.

  • a.
    Co-culture 1×10⁵ naïve CD4⁺ T cells with 1×10⁵ splenocytes as antigen presenting cells in round-bottom 96-well plates.
  • b.
    Add OVA peptide at 1 μg/mL to stimulate antigen-specific proliferation.
  • c.
    Add isolated saline or AC-treated isolated CD11b⁺GR1⁺ cells at ratios 1:1, 1:4, and 1:8 (MDSC:T cell).
  • d.
    Incubate for 72h at 37°C, 5% CO₂.
• 39.
  Cell proliferation analysis.

  • a.
    Centrifuge plate at 300 × g for 5 min.
  • b.
    Save supernatant in −80°C for future cytokine detection.
  • c.
    Stain cells for CD4 Va2 to identify OVA-specific T cells as described above (Figures 3B and 4B).
  • d.
    Analyze CTV dilution by flow cytometry to quantify T cell proliferation (Figure 3C).

CRITICAL: Titrate CTV concentration if you observe toxicity or weak resolution of proliferation peaks. Do not include serum or proteins in the staining step, as they can reduce dye uptake. Handle CTV and stained cells under low-light conditions to prevent photobleaching.

Figure 3.

Naïve CD4⁺ T Cell Isolation and Proliferation Assay

(A) Materials used for isolation of naïve CD4⁺ T cells from mouse splenocytes, including the EasySep™ Mouse Naïve CD4⁺ T Cell Isolation Kit, EasySep™ Magnet, and 5 mL round-bottom tubes.

(B) Purity assessment before and after isolation. CD4⁺ T cells were gated within CD3⁺ cells, and OVA-specific CD4⁺ T cells were identified by co-expression of TCR Vα2 and CD62L.

(C) Isolated CD4⁺ T cells were labeled with CellTrace™ Violet (CTV) and co-cultured with C57BL/6J splenocytes in the presence or absence of OVAp (1 μg/mL) and/or MDSCs (1:1 ratio). The first histogram shows an overlay of CTV dilution across conditions. Subsequent histograms display individual proliferation profiles.

Adoptive cell transfer and OVA immunization

Timing: 15 days

This step aims to assess antigen-specific adaptive immune responses in vivo and determine how MDSCs modulate these responses. Mice are immunized with the antigen of interest, and lymphoid organs are later collected to evaluate T and B cell activation, proliferation, and cytokine production upon antigen recall. The presence or adoptive transfer of MDSCs allows examination of their suppressive influence on the development and magnitude of antigen-specific immunity, providing functional insight into their regulatory role within the immune response.

• 40.
  Adoptive transfer.

  • a.
    Euthanize one naïve OT-II mouse using CO₂ euthanasia chamber followed by cervical dislocation as a secondary method.
  • b.
    Immediately harvest spleen under aseptic conditions and place in cold PBS.
    Prepare splenocyte single cell suspension as mentioned in item 2.
  • c.
    Inject 5 × 10⁶ total splenocytes intravenously into recipient mice (retro-orbital injection).
• 41.
  OVA immunization.

  • a.
    Wait 24 h post-transfer.

    • i.
      Immunize mice subcutaneously with 100 μg OVA adsorbed to Alum (1.5 mg Alum Imject).
  • b.
    Repeat immunization on day 7 (boost).
  • c.
    On day 15, euthanize mice and collect spleens for:

    • i.
      Flow cytometric phenotyping.
    • ii.
      In vitro restimulation with 2 μg/mL OVAp 323–339 peptide.
    • iii.
      Serum collection for OVA-specific IgG ELISA (see Mouse OVA-IgG titration ELISA).

Note: Each naïve OT-II mouse spleen yields around 50×10⁶ cells, therefore, calculate the amount of mice needed per experiment. To assess the suppressive effect of MDSCs in vivo, adoptively transfer 2×10⁶ isolated CD11b⁺GR1⁺ cells from either saline or alum-treated mice intravenously into recipient mice 24 h before immunization. Repeat all the immunization steps (Figure 4C).

CRITICAL: Randomize and blind investigators to group allocation when possible. Use consistent injection sites and volumes.

OVA-IgG titration ELISA

Timing: 1–2 days

This step aims to quantify OVA-specific IgG antibodies in the serum of immunized mice using an indirect ELISA. Serum samples are incubated on OVA-coated plates to allow antigen–antibody binding, followed by detection with enzyme-conjugated secondary antibodies specific for mouse IgG. The resulting colorimetric signal provides a quantitative measure of the humoral response, reflecting the magnitude of antigen-specific antibody production and the influence of experimental conditions such as MDSC-mediated immunosuppression.

• 42.
  Blood collection.

  • a.
    Anesthetize mice with 3%–5% isoflurane in an induction chamber.
  • b.
    Perform terminal blood collection via retro-orbital puncture using heparinized capillary tubes.
  • c.
    Centrifuge blood at 2,000 × g for 10 min at 4°C to separate plasma.
  • d.
    Aliquot plasma and store at −20°C until use.
• 43.
  Antibody detection - ELISA.

  • a.
    Coating the Plate.

    • i.
      Dilute OVA in carbonate buffer (0.05M pH 9.4–9.6) to 10 μg/mL.
    • ii.
      Add 100 μL per well. Cover and incubate for 18h at 4°C.
  • b.
    Blocking.

    • i.
      Wash plate 3× with PBS-T (PBS 0.05% Tween-20).
    • ii.
      Add 200 μL blocking buffer (PBS 1% BSA) per well. Incubate 1 h at 37°C.
  • c.
    Serum or plasma Titration.

    • i.
      Prepare 2-fold serial dilutions (start with 1:100 to 1:6400) in blocking buffer.
    • ii.
      Add 100 μL diluted serum to each well. Incubate 2 h at 37°C.
  • d.
    Detection.

    • i.
      Wash 3× with PBS-T.
    • ii.
      Dilute HRP-anti-mouse IgG to 1:10,000 in blocking buffer.
    • iii.
      Add 100 μL HRP-anti-mouse IgG per well. Incubate 1 h at 37°C.
  • e.
    Development.

    • i.
      Wash 3× with PBS-T.
    • ii.
      Prepare OPD (o-Phenylenediamine) working solution freshly before use by dissolving 1 OPD tablet in 10 mL of citrate buffer and adding 0.03% H₂O₂ (30 μL of 30% stock).
    • iii.
      Add 100 μL OPD solution per well. Develop ∼10–15 min in the dark.
    • iv.
      Stop with 50 μL stop solution (0.5M Sulfuric acid) per well.
  • f.
    Reading.

    • i.
      Read absorbance at 450 nm in plate reader.

CRITICAL: Ensure OVA is thoroughly dissolved and filter-sterilized before coating plates. Perform all incubations with gentle shaking for consistent signal development.

Note: Longer incubation time will lead to higher absorbance values, therefore, keep the development step fixed for all plates. Define IgG endpoint titer as the highest dilution with an OD value greater than the average OD of negative control wells plus 2× the standard deviation.

Western blot analysis of MDSCs

Timing: 2 days

This step aims to detect and compare IDO1 protein expression levels in CD11b⁺Gr1⁺ cells isolated from saline- or alum-treated mice. Following cell isolation, IDO1 expression is assessed by immunoblotting to determine changes in this immunoregulatory enzyme under different treatment conditions. Quantifying IDO1 levels provides insight into the mechanisms underlying alum-induced immunosuppression and the functional status of myeloid-derived suppressor cell populations.

• 44.
  Protein quantification.

  • a.
    Lyse 1×10⁶ isolated CD11b+GR1+ in 100 μL RIPA buffer + protease/phosphatase inhibitors on ice for 30 min.
  • b.
    Centrifuge lysate at 14,000 × g for 10 min at 4°C to remove debris.
  • c.
    Transfer sample to a new tube and keep it on ice.
  • d.
    Prepare a standard curve in the same buffer as your samples, typically ranging from 0 to 10 mg/mL in the following concentrations:

    • i.
      10, 5, 2.5, 1.25, 0.625, 0.31, 0.15, and 0mg/mL in duplicates.
  • e.
    Prepare Working Reagent (WR).

    • i.
      Mix Reagent A and Reagent B in a 50:1 ratio (e.g., 50 mL Reagent A + 1 mL Reagent B).
    • ii.
      Prepare enough WR for all wells (each well needs 200 μL of WR).
    • iii.
      Prepare fresh and use within 24 hours.
  • f.
    Plate Setup.

    • i.
      Pipette 10 μL of each standard or sample into wells of a 96-well plate (in duplicate or triplicate).
  • g.
    Add Working Reagent.

    • i.
      Add 200 μL of WR to each well using a multichannel pipette.
    • ii.
      Mix gently by pipetting up and down or tapping the plate gently.
  • h.
    Incubation.

    • i.
      Cover the plate with a lid or film to prevent evaporation.
    • ii.
      Incubate at 37°C for 30 minutes (alternatively, incubate at 25°C for 2 hours for more stable readings).
  • i.
    Reading the Plate.

    • i.
      Cool the plate to 25°C if incubated at 37°C.
    • ii.
      Measure absorbance at 562 nm using a microplate reader.
  • j.
    Data Analysis.

    • i.
      Generate a Standard Curve: Plot the average absorbance (y-axis) versus protein concentration (x-axis) for the BSA standards.
    • ii.
      Determine Sample Concentration: Use the standard curve to interpolate the protein concentrations of your unknown samples.
    • iii.
      Multiply by any dilution factor used during sample preparation.
• 45.
  Protein detection – Western Blotting.

  • a.
    Sample Preparation.

    • i.
      Normalize lysates to equal protein concentrations based on BCA results (20–40 μg per lane in 20 μL).
    • ii.
      Mix lysates 1:1 with 2× Laemmli buffer (final: 1×), including reducing agent.
    • iii.
      Boil samples at 95°C for 5 minutes (or 70°C for 10 minutes for sensitive proteins).
    • iv.
      Briefly spin down to collect condensation.
  • b.
    Gel Electrophoresis.

    • i.
      Assemble 12% acrylamide gel in an electrophoresis chamber and fill with running buffer.
    • ii.
      Load molecular weight marker and equal amounts of denatured protein per lane.
    • iii.
      Run the gel at 100 V through the stacking gel, then 120–150 V until the dye front reaches the bottom.
  • c.
    Protein Transfer.

    • i.
      Equilibrate gel and membrane in transfer buffer (5–10 minutes).
    • ii.
      Activate PVDF membrane in methanol for 1 minute, then rinse with water and transfer buffer.
    • iii.
      Assemble transfer sandwich (top to bottom): sponge-gel-membrane-sponge.
    • iv.
      Transfer proteins at 25 V for 7 min using TransBlot Turbo system.
  • d.
    Blocking.

    • i.
      Block the membrane in 1% BSA in TBS-T for 1 hour at 25°C with gentle shaking.
  • e.
    Primary Antibody Incubation.

    • i.
      Dilute primary antibody in blocking buffer or TBS-T.
    • ii.
      Incubate membrane with primary antibody for 18h at 4°C with gentle rocking.
    • iii.
      Antibody dilutions should be optimized (IDO1 1:1000, GAPDH 1:2000).
  • f.
    Washing.

    • i.
      Wash membrane 3× for 5–10 minutes in TBS-T at 25°C with shaking.
  • g.
    Secondary Antibody Incubation.

    • i.
      Incubate with appropriate HRP-conjugated secondary antibody (IDO1 – anti-mouse 1:1000, GAPDH anti-rat 1:1000) in blocking buffer or TBS-T.
    • ii.
      Incubate 1 hour at 25°C with shaking.
  • h.
    Washing.

    • i.
      Wash membrane 3× for 5–10 minutes in TBS-T at 25°C with shaking.
  • i.
    Detection.

    • i.
      Incubate membrane with ECL substrate (1:1 Reagent A and Reagent B, 1–5 minutes).
    • ii.
      Remove excess substrate and expose membrane using a BioRad ChemiDoc.
    • iii.
      Capture images at multiple exposure times to ensure linearity.
    • iv.
      Analyze band intensity using Bio-Rad Image Lab.

Note: To prevent issues using stripping buffers, we cut the membrane in two parts and incubated with the different primary antibodies.

Allogeneic pancreatic islet transplantation

Timing: 5–15 days

This step describes an allogeneic pancreatic islet transplantation model used to assess immune regulation and the suppressive function of MDSCs in vivo. Diabetes is chemically induced in recipient mice, through streptozotocin administration, to eliminate endogenous β-cell function. Purified pancreatic islets isolated from donor mice are then transplanted under the kidney capsule, where they can reestablish glycemic control. Graft success and function are monitored by measuring non-fasting blood glucose levels over time, providing a physiological readout of transplant acceptance or rejection.

• 46.
  Diabetes induction.

  • a.
    Prepare fresh STZ solution (10 mg/mL in sterile citrate buffer, pH 4.5) immediately before use.
  • b.
    Inject mice intraperitoneally with 240 mg/kg STZ.
  • c.
    Monitor blood glucose daily starting 48 h post-injection.
  • d.
    Consider mice diabetic when glucose >300 mg/dL on two consecutive days (Figure 5A).

CRITICAL: STZ is light-sensitive and unstable in solution. Prepare immediately before use and protect from light.

• 47.
  Diabetes induction.

  • a.
    Euthanize 8–10-week-old male Balb/c donor mice using approved methods.
  • b.
    Expose the common bile duct and cannulate with a 30G needle.
  • c.
    Gently inject 3–5 mL of cold collagenase solution into the pancreas via the bile duct.
  • d.
    Excise the inflated pancreas and digest at 37°C for 15 min.
  • e.
    Quench digestion with cold RPMI + 10% FBS and wash by centrifugation.
  • f.
    Purify islets using a density gradient (e.g., Histopaque) and hand-pick under a stereomicroscope.
  • g.
    Count and culture islets in complete RPMI at 37°C until transplantation (≤4 h post-isolation).

Note: Aim for 300–500 healthy, uniformly sized islets per transplant.

• 48.
  Islet transplantation under the kidney capsule.

  • a.
    Anesthetize recipient diabetic mice with 3%–5% isoflurane and maintain at 1%–2%.
  • b.
    Shave and disinfect the left flank.
  • c.
    Make a small incision to expose the kidney.
  • d.
    Carefully exteriorize the kidney using sterile forceps.
  • e.
    Load 300–500 islets in ≤10 μL volume into a Hamilton syringe.
  • f.
    Using fine forceps, create a small pocket under the kidney capsule.
  • g.
    Slowly inject the islets into the subcapsular space.
  • h.
    Gently return the kidney into the body cavity and close the incision using sutures or wound clips.
  • i.
    Place animals on a heating pad and monitor until full recovery.

CRITICAL: Avoid over-injection to prevent capsular rupture. Keep islet suspension volume as low as feasible.

• 49.
  Post-transplant monitoring.

  • a.
    Monitor mice daily for body weight, hydration, and surgical site condition.
  • b.
    Measure non-fasting blood glucose daily using tail vein sampling.
  • c.
    Define graft success as sustained blood glucose <200 mg/dL for >2 consecutive days.
  • d.
    Define graft failure as return to hyperglycemia (>300 mg/dL) after initial engraftment.
• 50.
  Quantification and data analysis.

  • a.
    Graft function.

    • i.
      Monitor non-fasting blood glucose daily using tail vein sampling.
    • ii.
      Define successful engraftment as blood glucose consistently <200 mg/dL for ≥2 consecutive days post-transplant.
    • iii.
      Define graft rejection or failure as sustained hyperglycemia >300 mg/dL on 2 consecutive days after a period of normoglycemia.
  • b.
    Kaplan–Meier survival analysis.

    • i.
      Record the day of graft rejection for each mouse (i.e., day when glucose permanently exceeds 300 mg/dL).
    • ii.
      Generate Kaplan–Meier survival curves using GraphPad Prism or similar statistical software.
    • iii.
      Compare groups using the Mantel–Cox (log-rank) test.
    • iv.
      Report median graft survival time, p-values, and number of animals per group (n).

CRITICAL: Ensure that all mice included in the analysis met baseline diabetes criteria (blood glucose >300 mg/dL for 2 days) before transplantation.

Figure 5.

Evaluating the Effects of Adjuvant Conditioning on Allogeneic and Human Immune Responses

(A) Experimental design for allogeneic pancreatic islet transplantation. WT mice treated with saline, or alum were rendered diabetic with streptozotocin (STZ) four days prior to transplantation of Balb/c islets. Graft survival was monitored by daily blood glucose measurements.

(B) Cutaneous delayed-type hypersensitivity (DTH) assay. WT mice were immunized subcutaneously with 20 × 10⁶ Balb/c splenocytes on the neck. Seven days later, mice were challenged with 20 × 10⁶ Balb/c splenocytes injected into the ear pinna. Ear swelling was measured at 2, 4, and 24 hours post-challenge, as also described by Zecher et al. (2009).³

(C) In vitro stimulation of human PBMCs. PBMCs (3 × 10⁵ per well) from five healthy donors were stimulated with alum (250 or 500 μg/mL) once, twice, or three times, followed by LPS (200 ng/mL) for 24 h in 96-well round-bottom plates. Supernatants were collected at designated time points for cytokine analysis. Image created with BioRender.

Cutaneous allogeneic hypersensitivity assay

Timing: 8 days

This step describes a cutaneous allogeneic hypersensitivity assay used to evaluate delayed-type hypersensitivity (DTH) responses as a measure of innate and T cell–mediated alloreactivity. Recipient mice are first sensitized with donor-derived splenocytes to prime the immune system against alloantigens. A subsequent recall challenge is performed by injecting donor cells into the ear pinna, where local inflammation develops in response to antigen recognition. Ear thickness is measured over time as a quantitative indicator of DTH magnitude, providing an in vivo readout of cellular immune activation and potential modulation by MDSCs or other immunosuppressive interventions.

• 51.
  Allogeneic sensitization - Day 0.

  • a.
    Prepare a suspension of 20 × 10⁶ Balb/c splenocytes in 100 μL sterile PBS.
  • b.
    Subcutaneously inject into the dorsal flank of C57BL/6 mice using a 26G needle.

Note: Ensure injection is placed in the subcutaneous space (Methods Video S1).

• 52.
  Recall challenge and measurement - Day 7.

  • a.
    Prepare a suspension of 20 × 10⁶ Balb/c splenocytes in 100 μL PBS.
  • b.
    Measure baseline ear thickness in both ears using a digital caliper (Figure 5C).
  • c.
    Inject 10 μL of the splenocyte suspension into the base of one ear pinna (Methods Video S2).

    • i.
      Optionally use isoflurane to reduce movement and stress.
  • d.
    At 2 h, 4 h, and 24 h post-challenge, measure ear thickness again at the same anatomical point.

CRITICAL: Perform all measurements blinded and by the same operator to reduce variability.

Human PBMC isolation and in vitro stimulation

Timing: 8–9 days

This step aims to model alum-induced immune modulation in vitro using human peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. PBMCs are stimulated with alum to mimic the conditioning effects observed in vivo, enabling assessment of how alum alters human immune cell activation. The response is evaluated by measuring the production of inflammatory cytokines and other immune mediators, providing insight into the capacity of alum to modulate innate immune signaling and shape subsequent adaptive immune responses.

• 53.
  PBMC isolation.

  • a.
    Dilute buffy coat 1:1 with PBS + 2% FBS + 4 mM EDTA.
  • b.
    Overlay 25 mL diluted blood onto 10 mL Lymphoprep in 50 mL tubes.
  • c.
    Centrifuge at 900 × g for 30 min at 25°C with no brake.
  • d.
    Collect PBMC layer carefully and wash twice with PBS + 2% FBS + 4 mM EDTA.
  • e.
    Lyse remaining RBCs by incubating with 10 mL ACK buffer for 10 min at 25°C.
  • f.
    Add 40mL pf PBS + 2% FBS + 4 mM EDTA and centrifuge at 400 × g for 5 min at 4°C.
• 54.
  Alum stimulation.

  • a.
    Resuspend cells in 5mL of complete RPMI medium and count cells.
  • b.
    Plate 3 × 10⁵ PBMCs per well in 96-well plates.
  • c.
    Stimulate with alum (250 or 500 μg/mL) once, twice, or three times at 24 h intervals (Figure 5D).
  • d.
    Between stimulations, replace media and collect supernatants for viability assays and storage at −80°C.
  • e.
    For final stimulation, add LPS (200 ng/mL) for 24 h.

    • i.
      Assess viability via LDH release assay including Triton X-100 positive and medium-only negative controls.

CRITICAL: Handle human samples under appropriate biosafety conditions. Use fresh reagents and sterile technique.

Expected outcomes

This collection of assays is designed to model the immunological impact of adjuvant conditioning (AC) in both mouse and human systems. In mice treated with alum, a significant expansion of CD11b⁺GR1⁺ myeloid-derived suppressor cells (MDSCs) is expected within the spleen.^1,3 These cells typically exhibit suppressive capacity in vitro, demonstrated by reduced CD4⁺ T cell proliferation in co-culture assays. Additionally, repeated alum injections lead to dampened antigen-specific responses following OVA immunization,¹ reflected by reduced splenocyte proliferation and lower OVA-specific IgG titers measured by ELISA.

Flow cytometric analysis of lymphoid organs should reveal increased frequencies of MDSCs, decreased effector CD4⁺ T cell activity, and potentially enhanced regulatory populations, depending on the readouts used. In adoptive transfer models, MDSCs isolated from alum-treated mice consistently suppress the host’s T cell responses in vivo. Pancreatic islet transplantation in alum-conditioned or MDSC-recipient mice typically results in prolonged graft survival, with normoglycemia (<200 mg/dL) maintained for several days or weeks post-transplant.^1,3 In contrast, control mice rapidly reject the graft.

In the human system, PBMCs repeatedly stimulated with alum exhibit a blunted cytokine response to subsequent LPS challenge, as measured by ELISA or bead-based multiplex assays. This suggests functional adaptation or innate immune tolerance. Viability remains acceptable across most donors, though cytotoxicity may increase with repeated alum exposure or donor variability. Overall, these protocols consistently demonstrate that adjuvant conditioning induces an immunosuppressive state across both murine and human models, characterized by suppressed inflammatory cytokine production, T cell function, and alloreactivity.

Limitations

While these protocols provide a robust model to study the immunomodulatory effects of adjuvant conditioning, there are important limitations to consider. Additionally, although the in vivo experiments demonstrate systemic immune suppression, they rely on surrogate readouts (e.g., graft survival, ear swelling), which do not fully capture cellular reprogramming or long-term tolerance induction.

In the human PBMC model, donor variability poses a significant limitation, particularly in cytokine output and sensitivity to alum and LPS. Moreover, in vitro culture conditions cannot fully recapitulate the complex in vivo microenvironment, limiting translational conclusions. Finally, the reliance on endpoint assays such as ELISA or flow cytometry restricts the temporal resolution of immune activation dynamics and does not include transcriptomic or epigenetic profiling, which could reveal deeper mechanistic insight into the effects of adjuvant exposure.

Troubleshooting

Problem 1

Inconsistent immunosuppressive response after alum conditioning (steps 1–5).

In some animals, the expected reduction in inflammatory cytokines or increase in MDSC frequency may not be observed.

Potential solution

Check the homogeneity of the alum suspension prior to each injection—alum tends to settle quickly and requires thorough vortexing. Ensure that injections are spaced consistently (every other day) and are delivered intraperitoneally with minimal variation in volume and location. Additionally, verify that the batch of alum has not expired or changed formulation.

Problem 2

Weak T cell proliferation or absence of suppression in adoptive transfer assays (steps 33–41).

After immunization and MDSC transfer, no significant suppression of T cell proliferation is observed in some mice.

Potential solution

Confirm that MDSCs are isolated with high purity (>90%) and viability. Perform the adoptive transfer exactly 24 hours prior to OVA immunization to allow time for in vivo engraftment. Additionally, confirm OVA peptide adsorption to adjuvant and titrate if necessary to optimize antigen presentation.

Problem 3

Cell death or poor yield during MDSC isolation and culture (steps 33 and 34).

MDSCs show high death rates during culture or do not respond to LPS stimulation.

Potential solution

Reduce centrifugation speed and duration to minimize mechanical stress during isolation. Ensure the LPS is freshly prepared and used at a validated concentration (e.g., 200 ng/mL). Maintain sterility and confirm correct 2-mercaptoethanol concentration in the culture medium (0.05 mM).

Problem 4

High background staining in flow cytometry (steps 16–32).

Flow cytometry shows high nonspecific signal, especially in intracellular staining.

Potential solution

Titrate all antibodies individually and include fluorescence-minus-one (FMO) controls. Increase wash steps and ensure thorough blocking with Fc receptor blockers. Use viability dyes to exclude dead cells that often bind antibodies nonspecifically.

Problem 5

Failure to observe suppression in T cell proliferation assay (steps 35–39).

Even with MDSC co-culture, CD4⁺ T cells continue to proliferate robustly.

Potential solution

Check the labeling efficiency and consistency of the CellTrace Violet (CTV) dye. Inconsistent dye loading can obscure suppression. Test multiple MDSC:T cell ratios (1:1, 1:4, 1:8) and confirm that OVAp concentration is optimized for robust T cell activation. An alternative would be to test total T cell activation using CD3 and CD28 beads instead of OVAp.

Problem 6

No IDO1 band in Western blot (steps 44 and 45).

Expected IDO1 protein band is absent.

Potential solution

Verify total protein concentration using a BCA assay before gel loading and consider loading more protein on the gel. Optimize transfer time, antibody concentration, and ensure the primary antibody is validated for Western blot. Use a positive control lysate if needed.

Problem 7

PBMC viability loss after alum stimulation (steps 53 and 54).

Human PBMCs show elevated LDH release after stimulation.

Potential solution

Alum dosage may need to be titrated for each donor batch. Avoid repeated pipetting and use gentle media exchanges. Include viability controls using known cytotoxic agents and vehicle-only wells.

Problem 8

Islet graft rejection or no normalization of glucose (steps 46–50).

Mice remain hyperglycemic after transplant or quickly revert to hyperglycemia.

Potential solution

Verify islet count (aim for 300–500) and minimize islet ischemia by transplanting within 4 h of isolation. Practice surgical technique to prevent islet loss due to capsular rupture or leakage. Ensure diabetic status pre-transplant was confirmed (glucose >300 mg/dL).

Problem 9

No ear swelling in cutaneous hypersensitivity assay (steps 51–54).

Sensitized mice fail to mount a response after ear challenge.

Potential solution

Ensure proper sensitization with viable Balb/c splenocytes 7 days prior. Confirm injection of 100 μL cell suspension into the base of the ear pinna and measure ear thickness at a consistent point. Consider using positive controls and verifying splenocyte viability.

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Alex G. Cuenca (alex.cuenca@childrens.harvard.edu).

Technical contact

Technical questions can also be addressed to the technical contact, Thais Boccia (thaisbc@mit.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

Data acquired specifically for this study are available within the article itself and the supplemental materials. Experimental protocols and additional details regarding methods employed in this study will be made available through reasonable request with the corresponding author.

Acknowledgments

This work was supported by the American Pediatric Surgical Association Jay Grosfeld Scholar Grant, Society of University Surgeons Junior Faculty Award, Hardy Hendren Faculty Development Fund at Boston Children’s Hospital, and the Junior Translational Investigator Service Award from the Translational Research Program at Boston Children’s Hospital. BioRender was used to create the graphical abstract.

Author contributions

Conceptualization, T.B. and A.G.C.; methodology, T.B., M.D.V.d.S., V.F., and W.P.; writing – original draft, T.B.; writing – review and editing, T.B., V.F., M.D.V.d.S., M.S.R., W.P., and A.G.C.; funding acquisition, A.G.C.; resources, M.S.R., V.F., and A.G.C.; supervision, A.G.C.

Declaration of interests

The authors declare no competing interests.