A Neonatal Intravenous Transplant Model for Assessment of Early-Life Immune Activities Against Cancer Cells

Summary

This protocol demonstrates temporal vein injection for intravenous delivery of pediatric cancer cells to neonatal mice, enabling precise, reproducible modeling of early-life immune responses against cancer. The method enables assessment of the impact of interventions, such as immunomodulation or therapy, when applied during the neonatal period.

Abstract

More than half of all pediatric cancers are initiated in utero, with progression towards overt disease continuing through the neonatal period. While immunotherapies now offer new treatment options for many children with cancer, how early-life immunity influences cancer progression and therapy response remains incompletely understood. Addressing this gap will require evaluation of therapeutic interventions during the neonatal period, when immune responses differ fundamentally from those in later childhood and adulthood.

Here, we describe a neonatal transplantation model that integrates with bioluminescent in vivo imaging to investigate the impact of early-life immune modulation on pediatric cancers.

Temporal vein injection of luciferase-tagged cells derived from two in utero-initiated cancers achieved reproducible tumour engraftment in 1–2-day-old syngeneic recipients, with no procedure-related mortality and high procedural success as confirmed by bioluminescent imaging. Using infection-driven immune modulation as an experimental variable, we then show that intraperitoneal infection with murine cytomegalovirus five days after transplantation results in a marked reduction in the burden of acute lymphoblastic leukemia (ALL), but not neuroblastoma (NB). This platform enables the study of cancer-specific differences in response to early-life immune modulation and underscores the importance of tumor context when modeling immune–cancer interactions in early life.

Collectively, this methodology provides a versatile and highly reproducible platform for interrogating neonatal immune mechanisms relevant to cancer immunosurveillance, infection-driven immune modulation, and therapeutic intervention. Beyond ALL and NB, this approach can be adapted to additional pediatric malignancies and immunomodulatory strategies, supporting mechanistic studies and preclinical evaluation of early-life cancer interventions.

Introduction

Recent advances in immunotherapy have markedly enhanced outcomes for certain cancer patients, yet their optimal application in young children necessitates a thorough understanding of how the developing immune system identifies and eradicates cancer cells. While approximately 50% of childhood cancers originate in utero, including B-cell acute lymphoblastic leukemia (B-ALL) and NB, growing evidence suggests that postnatal events critically influence disease evolution and clinical outcome^1,2.

Neonatal immune responses differ quantitatively and qualitatively from those of adults³. In the absence of fully developed adaptive immunity, protection of neonates relies heavily on maternal antibodies and innate immune cells. These innate effectors, however, are functionally less efficient than their adult counterparts due to cell-intrinsic hypo-responsiveness and enhanced activation of immunosuppressive pathways, leaving neonates particularly susceptible to infection^4,5,6. However, the impact of these characteristics on the ability of the immature immune system to mount effective anti-cancer responses remains poorly understood.

Modeling anti-cancer immunity in early life requires the precise and reproducible delivery of cancer cells and immune modulators or pathogens during the short postnatal window, when immune function is limited. Temporal vein injection is a neonatal intravenous (IV) delivery method that best meets these requirements and offers high procedural success, low mortality, minimal handling stress, and compatibility with sequential interventions, such as infection challenge or therapeutic administration^7,8,9,10.

In this study, we built upon and modified previously validated neonatal intravenous delivery techniques and the versatility of bioluminescent in vivo imaging to describe a transplant model to study the impact of early-life infection on the progression of pediatric cancer cells. This approach enables the intravenous delivery of luciferase-tagged B-ALL or NB cells to 1–2-day-old pups, followed by immune modulation through intraperitoneal (i.p.) administration of murine cytomegalovirus (mCMV) on postnatal day 7 (P7). Cancer progression was subsequently evaluated 10 days post-infection (P17).

Although access to the temporal vein is restricted to a limited postnatal window (P0–P3), this methodology enhances reproducibility and experimental control, offering a versatile platform to investigate the influence of early-life immune mechanisms on cancer immunosurveillance, infection–cancer interactions, and responses to immunotherapy. Additionally, this approach supports the efficient homing of transplanted cells, thereby enabling the reproducible establishment of disease models for mechanistic investigations.

Protocol

All procedures (Figure 1) were conducted in accordance with the Canadian Council on Animal Care guidelines and were approved by the University of British Columbia Animal Care Committee (protocol A23-0169).

1. Generation of luciferase-tagged leukemic cells

1. Isolation of Leukemic Cells from leukemic Eµ-Ret mice
   1. Anesthetize mice in a 1 L induction chamber with 5 % isoflurane and 1 L·min^-1 oxygen until a deep surgical plane is achieved (loss of righting reflex and slow, regular respiration).
   2. Once deep anesthesia is confirmed, discontinue isoflurane and administer carbon dioxide (CO₂) at a high flow rate (3–4 times the standard gradual displacement rate; e.g., 6-8 L·min for a 10 L chamber) without transferring the animal between chambers. Maintain CO₂ exposure for at least 5 min after respiration ceases to ensure death.
   3. Vent and scavenge the waste gases before opening the chamber.
   4. Confirm death by absence of heartbeat and respiration, then perform cervical dislocation as a secondary physical method in accordance with institutional.
   5. Transfer the carcass to a biosafety cabinet, reconfirm death, record sex and body weight, and spray the exterior with 70 % ethanol.
   6. Make a midline abdominal incision, reflect the skin and peritoneal wall, and excise the spleen into 5 mL sterile PBS.
   7. Dissect tibiae and femora from both legs, removing excess tissue, and excise the spleen under sterile conditions. Immediately transfer the spleen into 5 mL sterile, ice-cold Phosphate Buffered Saline (PBS) solution to preserve cell viability.
   8. Process the spleen.
      1. Homogenize spleen tissue in 1 mL of 1× red blood cell (RBC) lysis buffer (use 2 mL for spleens > 200 mg). Incubate for 2–3 min at room temperature.
      2. Quench lysis by adding 10 mL PBS + 2 % fetal bovine serum (FBS) (FACS buffer), filter through a 40 µm strainer, and centrifuge at 350 × g for 5 min at 4 °C.
      3. Resuspend the spleen pellet in 5 mL FACS buffer.
   9. Process bone marrow.
      1. Excise both epiphyses and flush each bone with 1 mL FACS buffer using a 1 mL syringe fitted with a 29-gauge needle into a 15 mL tube.
      2. Centrifuge the cell suspension, discard the supernatant and resuspend cells in 1 mL of 1× RBC lysis buffer, incubate for 2 min at RT, the quench lysis by adding 10 mL FACS buffer, filter through a 40 µm strainer, and centrifuge at 350 × g for 5 min at 4 °C.
      3. Discard the supernatant and resuspend the BM pellet in 1 mL FACS buffer by pipetting up and down.
      4. Vortex gently and filter through a 40 µm strainer if needed.
      5. Resuspend 0.5 × 10^6 leukemic cells in 200 µL cell culture media (e.g. Dulbecco's Modified Eagle Medium (DMEM)) supplemented with 20 % FBS and interleukin-7 (IL-7; 250 pg·mL^-1).
         Pause Point: Keep cell suspensions on ice for up to 2 h before staining.
2. Lentiviral transduction of leukemic cells:
   1. Seed murine bone marrow–derived stromal cells at 1 × 10^5 cells per well in a 24-well plate 24 h prior to transduction to allow adherence and achieve 70–80% confluency.
   2. Plate leukemic cells (0.5 × 10^6 cells in a final volume of 200 µL) in a non–tissue culture treated 24-well plate.
   3. Add a self-inactivating (SIN) lentiviral vector encoding a double-fusion reporter consisting of click beetle red (CBR) luciferase and enhanced green fluorescent protein (eGFP), driven by a constitutive ubiquitin promoter, with the multiplicity of infection (MOI): 10–20, viral titer: ≥ 1 × 10^8 TU·mL^-1 (transducing units per milliliter) in final transduction volume of 200 µL together with polybrene (8 µg·mL^-1; Sigma, H9268-5G).
      CAUTION: Polybrene is cytotoxic at high concentrations. Do not exceed 8 µg·mL^-1. Handle lentivirus under Biosafety Level 2 (BSL-2) containment.
   4. Seal the plate with parafilm and centrifuge at 1,200 × g for 20 min at room temperature (RT).
   5. Following spinoculation, transfer transduced leukemic cells onto stromal monolayer and culture for 24 h at 37 °C, 5% CO₂ in DMEM + 20 % FBS + IL-7 (250 pg·mL^-1) to support cell survival and reporter expression.
      NOTE: Antibiotics may be added after 24 h if required but should be avoided during viral transduction.
   6. After 24 h, collect non-adherent leukemic cells and wash twice with sterile phosphate-buffered saline (PBS), 350 × g for 20 minutes at 4 °C, to remove residual virus and polybrene.
   7. Resuspend cells in sterile PBS at 1 × 10^7 cells·mL^-1 for transplantation.
      ​Pause Point: After washing, cells can be maintained under the same culture condition up to 48 h before transplantation if needed.
3. Transplantation into adult NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) mice
   1. Use 4–6-week-old NSG mice as recipient.
   2. Warm mouse for 5 min using a warming pad or heat lamp to dilate the tail veins.
   3. Restrain mouse using an acrylic restrainer.
   4. Inject 1 × 10^6 cells in 100 µL sterile PBS via lateral tail vein using 29-gauge insulin syringe. No anesthesia required (unless required by institutional guidelines)
   5. Apply gentle pressure to the injection site to prevent bleeding.
      CAUTION: Avoid extravasation. Successful intravenous injection results in no visible swelling.
   6. Monitor mice twice weekly for disease development.
4. Sorting GFP⁺ leukemic cells
   1. Upon disease onset, harvest BM or spleen from leukemic NSG mice.
   2. Prepare a single-cell suspension as described above.
   3. Stain cells with viability dye (7-AAD).
   4. Use Fluorescence-Activated Cell Sorting (FACS) for sorting GFP+ leukemic cells.
   5. For sorting use high-speed cell sorter and follow the gating strategy of Forward scatter (FSC) vs side scatter (SSC) → exclude debris, FSC-A vs FSC-H → select singlets, Viability dye negative → select live cells, GFP+ gate → isolate reporter-positive leukemic cells.
   6. Collect sorted cells into tubes containing DMEM + 20 % FBS. Target post-sort purity: ≥ 95 % GFP+ cells.
      Pause Point: Sorted cells may be kept on ice up to 2 h before cryopreservation.
5. Cryopreservation of sorted cells
   1. Resuspend sorted cells at 5 × 10^6 cells/mL in freezing medium containing 90 % FBS and 10 % dimethyl sulfoxide (DMSO).
   2. Aliquot 1 mL per cryovial and place vials in isopropanol-based freezing container and Store at −80 °C overnight (12–18 h).
   3. Transfer vials to liquid nitrogen (LN₂) for long-term storage.
      CAUTION: DMSO is toxic and readily absorbed through the skin. Handle with gloves and work quickly.
      Pause Point: Cells may be stored long-term in liquid nitrogen until use in neonatal transplantation experiments.

2. Generation of luciferase-tagged NB cells

NOTE: TH-MYCN transgenic mice develop spontaneous NB tumors and were maintained under approved institutional animal care protocols. Lentiviral vector used is the same as described in Section 1.2.

1. Tumor Harvest and Tissue Dissociation
   1. Euthanize tumor-bearing TH-MYCN mice according to approved animal care guidelines.
   2. Harvest subcutaneous or abdominal NB tumors under sterile conditions.
   3. Weigh tumor tissue and mince into ~1–2 mm fragments using sterile scissors.
   4. Transfer tissue into approximately 5 mL digestion buffer per 0.5 g tumor tissue in a 15 mL conical tube, consisting of DMEM, Deoxyribonuclease I (DNase I; 50–100 U·mL^-1), and collagenase-based enzyme blend (10 µg·mL^-1 final concentration).
   5. Incubate at 37 °C in a shaking incubator at 150 rpm for 30 min.
      CAUTION: Enzymatic digestion reagents may cause skin and eye irritation. Wear gloves and eye protection.
   6. After digestion, gently pipette up and down to further dissociate tissue. Filter suspension through a 70 µm cell strainer into a fresh tube.
   7. Centrifuge at 300 × g for 5 min at room temperature (20–22 °C).
   8. Discard supernatant and resuspend pellet in DMEM supplemented with 10% FBS.
   9. Perform RBC lysis using RBS lysis buffer as previously described in Section 1.1.
   10. Centrifuge at 300 × g for 5 min and resuspend pellet in 10 mL high-glucose DMEM supplemented with 10% FBS and 50 µM 2-mercaptoethanol.
       CAUTION: 2-mercaptoethanol is toxic and volatile. Perform handling in a chemical fume hood.
       Pause Point: Cells may be kept on ice for up to 1 h before transduction.
2. Lentiviral Transduction
   1. Pellet 2 × 10^6 NB cells at 300 × g for 5 min and remove supernatant completely.
   2. Resuspend cell pellet directly in 14 µL concentrated lentiviral vector (same vector described in Section 1.2; viral titer ≥ 1 × 10^8 TU·mL-1, approximate MOI: 10–20).
   3. Incubate cell–virus mixture for 5 min at 37 °C in a humidified incubator (% CO₂).
   4. Add 200 µL high-glucose DMEM + 10% FBS and continue incubation for 2 h at 37 °C, 5% CO₂.
      CAUTION: Lentiviral vectors must be handled under BSL 2 conditions.
   5. Wash transduced cells four times with high-glucose DMEM to remove residual virus.
   6. Count viable cells using trypan blue exclusion.
      IMPORTANT: Keep matrix preparation on ice at all times to prevent premature gelation.
3. Subcutaneous Implantation into NSG Mice
   1. Resuspend cells at 5 × 10^6 cells per 100 µL in a cold basement membrane matrix preparation (growth factor-reduced extracellular matrix).
   2. Anesthetize 6–8-week-old NSG mice (see Section 1.1) using inhaled isoflurane (2–3% in oxygen).
   3. Load cell–matrix mixture into a pre-chilled 1 mL syringe fitted with a 27-gauge needle.
   4. Inject 5 × 10^6 cells in 100 µL total volume subcutaneously into the flank region.
   5. Monitor mice until full recovery from anesthesia.
      NOTE: Inject immediately after mixing with the matrix to prevent solidification inside the syringe.
   6. Allow tumors to grow to ~1 cm in diameter.
4. Cell sorting
   1. Harvest tumors and generate single-cell suspensions as described in Sections 2.1–2.2.
   2. Enrich luciferase-tagged cells by FACS based on GFP expression, following the gating strategy described in Section 1.4.
   3. Perform two sequential rounds of sorting to achieve ≥ 95% GFP-positive purity.
5. Cryopreservation
   1. Cryopreserve sorted cells as described in Section 1.5.
      Pause Point: Cells can be stored long-term in liquid nitrogen for future transplantation experiments.

3. Prepare the cell suspension for temporal vein injection

1. Thaw cryopreserved luciferase-tagged leukemic cells or NB cells rapidly in a 37 °C water bath until a small ice crystal remains.
2. Immediately disinfect the vial exterior with70 % ethanol, then transfer contents into 9 mL of pre-warmed DMEM + 10 % FBS medium in a 15 mL conical tube.
3. Centrifuge at 300 × g for 5 min at 4 °C, aspirate supernatant, and resuspend the pellet in sterile, ice-cold PBS.
4. Determine viability using trypan blue exclusion and a hemocytometer (or automated cell counter). Use only suspensions with ≥ 80 % viability.
5. Pass the cell suspension through a 40 µm cell strainer to remove clumps.
   CAUTION: Cell clumps increase failed injections and tissue injury.
6. Adjust concentration to prepare 20 µL per neonate containing 1 × 10^4 leukemic cells (for leukemic transplantation), or 5 × 10^4 NB cells (for NB transplantation).
7. Keep the cell suspension on ice and gently mix immediately before loading the syringe to maintain homogeneity.
   Pause Point: Keep cells on ice for up to 2 h before injection if viability remains ≥ 80%.

4. Temporal vein injection (P1–P2 pups)

NOTE: Neonatal temporal vein injection can be performed without anesthesia. However, brief hypothermic immobilization is commonly used to reduce movement and improve injection accuracy. The use of hypothermic immobilization is not permitted by some institutional animal care committees - investigators must consult with their relevant institutional committee before performing this procedure. If approved by your institution, the following steps are effective for hypothermic immobilization.

1. Prepare a small container lined with a sterile napkin and pre-chill it by placing it on ice.
2. Place pups (postnatal day 1–2; P1–P2) on the napkin inside the container for hypothermic immobilization for 2–3 min until spontaneous movement is markedly reduced. 
   IMPORTANT: Do not allow pups to contact ice directly (use the container and napkin barrier at all times).
3. Load 60 µL of the cell suspension into a 100 µL glass syringe and attach a 30-gauge needle. Expel the suspension back into the tube to fill the dead volume of the syringe and needle. Once the dead volume is primed, aspirate 20 µL of cell suspension for injection, ensuring that no air bubbles are present. Use BALB/c and TH-MYCN mice as recipients of leukemic and NB cells, respectively.
4. Prepare a transilluminated stage by placing a cold light source beneath a plexiglass platform.
5. Wear magnifying eyewear (or use a stereomicroscope) to visualize the injection site.
6. Place a sterile napkin with a central hole on the stage and position the pup so the temporal vein is visible.
7. Place a small piece of modeling clay (playdough) adjacent to the pup’s head to help stabilize the needle during injection.
8. Insert the needle bevel-up into the visible temporal vein and slowly inject the cell suspension, observing vascular filling to confirm successful intravenous delivery.
9. After injection, apply gentle pressure to the injection site using a sterile cotton swab. Place the pup on a warmed recovery surface (approximately 37 °C, e.g., heating pad on low with a towel barrier) until normal movement and pink coloration return.
10. Monitor the pup for 5–10 min after recovery, then return it to the dam.
    CAUTION: Avoid overheating during recovery. Use a barrier layer and monitor continuously.
11. Rinse the syringe immediately with PBS, then with a syringe cleaning solution, and finish with 70% ethanol.

5. Virus preparation and infection

1. Perform all procedures involving murine cytomegalovirus (mCMV) under BSL-2 containment in accordance with institutional biosafety and animal care approvals.
2. Handle virus stocks in a certified Class II biological safety cabinet and perform animal infections in designated BSL-2 procedure areas.
3. Conduct the virus preparation and handling in a certified Class II biological safety cabinet, and perform the animal infections in designated BSL-2 procedure areas.
4. Wear appropriate PPE, including a laboratory coat or disposable gown, gloves, and eye protection. Treat all virus-containing materials (syringes, bedding, cages, and surfaces) as potentially infectious.
5. Minimize sharps use and dispose of needles immediately in approved biohazard sharps containers. Decontaminate work surfaces and equipment after use. Dispose of all solid and liquid waste as biohazardous waste according to institutional procedures.
6. Infect mice at P7 (5–6 days after leukemic cell transplantation).
7. Thaw a virus aliquot on ice, mix gently by inversion, and keep on ice during preparation. Avoid repeated freeze–thaw cycles (use single-use aliquots).
8. Prepare mCMV at 2 × 10^5 plaque-forming units (PFU)/mL in PBS and inject pups intraperitoneally (i.p.) with 1 × 10^4 PFU in 50 µL PBS using a 30-gauge needle. Inject into the lower right quadrant of the abdomen at a shallow angle to avoid abdominal organs.
   CAUTION: mCMV is infectious (BSL-2). Use appropriate containment and decontamination.
9. Record animal identification, age, experimental group, virus dose, volume, and injection details.

6. Bioluminescence imaging (BLI)

NOTE: Perform BLI at P7 (5–6 days after cell injection and prior to infection) and at P17 (10 days post-infection) as described below.

1. Dissolve D-Luciferin in PBS at 10 µg·µL^-1. Filter-sterilize using a 0.22 µm filter, aliquot, and store at −20 °C protected from light.
   CAUTION: D-luciferin is light-sensitive. Minimize light exposure during preparation and use.
2. Inject D-luciferin i.p. at 50 µL per pup using a 30-gauge needle.
3. Anesthetize pups with isoflurane (induction 3–4 %, maintenance 1.5–2 %) in oxygen (0.8–1.0 L·min^-1). Maintain body temperature on a warmed imaging stage (approximately 37 °C).
   CAUTION: Isoflurane is a volatile anesthetic. Use an approved vaporizer and scavenging system.
4. Place anesthetized pups in a small-animal imaging system (instrument details provided in Table of Materials).
5. Acquire bioluminescent and X-ray images using consistent exposure settings 10 minutes after D-luciferin injection (BLI settings: exposure time 120 s for P7 images and 30 s for P17, binning B4 for P7 and B4 for P17 images, field of view (FOV) 25, f-stop 1.2, object height 1.5 and X-ray settings: resolution high, power high).
   Pause Point: Store images files immediately after imaging; analysis can be completed later.
6. Analyze images using appropriate BLI imaging software by defining regions of interest (ROIs) encompassing the whole body of each mouse and quantifying emitted light as photons·s⁻¹·cm⁻²·sr⁻¹.
7. Export quantified ROI values for downstream statistical analysis.

7. Harvesting leukemic cells from cell transplanted mice

1. Harvest leukemic cells and generate single-cell suspensions as described in Section 1.1.

8. Flow cytometry staining and acquisition

1. Count cells and centrifuge suspensions at 300 × g for 5 min at 4 °C and resuspend at 1 × 10^7 cells·mL^-1 in FACS buffer.
2. Transfer 100 µL (1 × 10^6 cells) into polystyrene flow cytometry tubes.
3. Add Fc receptor blocking antibody (anti-CD16/32; 1 µL per tube) and incubate for 5 min at room temperature.
4. Prepare antibody master mix in FACS buffer and add to each tube CD19-FITC, BP1-PE, B220-APC, and lineage negative markers (TER119-PerCP, CD3-PerCP, CD11b-PerCP), 1 µL of each antibody per 1 x 10^6 cells in 100 µL FACS buffer.
5. Incubate for 20 min at room temperature in the dark.
6. Wash with 5 mL FACS buffer, centrifuge, and resuspend in 200 µL FACS buffer containing 7-AAD (3 µL) and counting beads (6 µL; 6,000 beads).
7. Prepare single-stained compensation controls using antibody capture beads or cells and acquire controls alongside experimental samples.
8. Acquire samples on a flow cytometer using standard FITC/PE/APC/PerCP detector settings and a medium flow rate. Collect events until 1,000 beads are acquired.
9. Export FCS files and analyze using flow cytometry analysis software (listed in Table of Materials) using the established gating strategy (Figure 2D).
10. Calculate absolute cell numbers per spleen or bone marrow using counting beads by multiplying the number of target events acquired at 1,000 beads by a scaling factor of 6 (6,000 beads added), and normalizing to total organ cellularity based on the fraction of cells stained (1 × 10^6 cells).

9. Plotting graphs and statistical analysis

NOTE: Generate graphs and perform statistical analyses for BLI and flow cytometry data using a statistical software package (listed in Table of Materials).

1. Present data as mean ± standard error of the mean (SEM).
   NOTE: No animal was excluded from this study.
2. For BLI, compare PBS and mCMV groups separately at P7 and P17 using unpaired Mann–Whitney tests, with Holm–Šídák correction applied to adjust for multiple comparisons across time points.
3. Analyze flow cytometry outcomes using a two-tailed unpaired Mann–Whitney test.
4. Define statistical significance as p < 0.05.

Results

Temporal vein injection enables efficient neonatal engraftment of leukemia and neuroblastoma cells
To model the impact of early-life infection on pediatric cancer progression, luciferase-tagged B-ALL cells derived from leukemic adult Eµ-Ret mice or NB cells derived from moribund Th-Mycn mice were intravenously delivered via the temporal vein into 1–2-day-old syngeneic pups. Successful engraftment was confirmed five days post-transplantation using BLI and X-ray imaging for both leukemia (

Discussion

Understanding how early-life immune development influences cancer progression is essential for designing age-appropriate immunomodulatory strategies. In this study, we describe a reproducible neonatal transplantation platform that enables precise temporal control of cancer cell engraftment and immune modulation during a critical window of postnatal immune immaturity. By combining temporal vein–mediated intravenous delivery with BLI, this model allows systematic interrogation of how early-life infection shapes pedia...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100µL, Microliter Syringe, Luer Tip	Hamilton	80601	-
30 G Needle	BD	305106	-
7-AAD	Biolegend	420404	-
Ami-X imaging system	Spectral Instruments Imaging	-	-
anti-mouse B220- APC	Biolegend	103212	-
anti-mouse CD19-FITC	Biolegend	152404	-
anti-mouse CD3ε- PerCP	Biolegend	100326	-
anti-mouse Ly-51-PE	Biolegend	108308	Also called BP1
anti-mouse TER119- PerCP	Biolegend	116226	-
anti-mouse/human CD11b- PerCP	Biolegend	101230	-
Aura software	Spectral Instruments Imaging	-	version 4.0.8
BD Accuri C6	BD	-	-
BD Fortessa X-20	BD	-	-
Cleaning solution	Hamilton	18310	-
CountBright Absolute Counting Beads	Invitrogen by Thermo Fisher Scientific	2770930	6 µL contains 6000 beads
D-Luciferin	GoldBio	eLUCK-1G	-
DMEM	Gibco	11965092	-
DNase I	Sigma	1.13E+10	-
Falcon 40µm Cell Strainer, Blue, Sterile, Individually Packaged	FALCON	352340	-
FBS	Gibco	A5256701	-
FlowJo software	BD	-	v10.8
GeltrexTM	Thermo Fisher Scientific	A1413202	-
GraphPad Prism software	Dotmatics	-	v10.4.1
LE.5209 Cold light source	Euromex	-	-
Liberase™ TM Research Grade	Sigma	5.4E+09	-
NSG mice	Jackson Labrotary	5557	NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ
Polybrene	Sigma	H9268-5G	-
Purified anti-mouse CD16/32 Antibody	Biolegend	101301	-
RBC Lysis Buffer (10X)	Biolegend	420301	-