Materials and Reagents

Micropipette tips (Rainin, catalog numbers: 17005860 [20 µl]; 30389240 [200 µl]; 30389213 [1,000 µl])
Corning^® non-treated culture dishes (D × H 100 mm × 20 mm) (Corning, catalog number: 430591 )
Syringe (3 ml) (Fisher Scientific, catalog number: 14-823-436 )
Conical tube (50 ml) (Thermo Scientific, catalog number: 339653 )
Cell strainer Nylon (100 µm) (BD Falcon, catalog number: 352360 )
Aluminum foil
Integra^TM Miltex^TM Sterile Surgical Blades (Fisher Scientific, catalog number: 12-460-444 )
Microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-137 )
Falcon^TM Round-Bottom Polystyrene Tubes (Flow tubes) (Corning, catalog number: 352054 )
ACK lysing buffer (Quality Biologicals, catalog number: 118-156-101 )
Dulbecco’s Modified Essential Medium (DMEM) high glucose (MilliporeSigma, catalog number: D5796 )
Heat Inactivated FBS (HI-FBS) (Fisher Scientific, catalog number: SH30109.03HI )
Collagenase Type 4 (Worthington Biochemical Corporation, catalog number: CLS4 )
Hyaluronidase (MP Biomedicals, catalog number: 0 2151272 )
Phosphate buffered saline (PBS) (Corning Cellgro, catalog number: 21-040-CV )
OneComp eBeads (eBioscience, catalog number: 01-1111-42 )
eBioscience^TM Fixation/Permeabilization Concentrate (Thermo Scientific, catalog number: 00-5123-43 )
eBioscience^TM Fixation/Permeabilization Diluent (Thermo Scientific, catalog number: 00-5223-56 )
Anti CD16/32 antibody (Fc blocker) (BioLegend, catalog number: 101302 ), dilution 1:100
Zombie Aqua (Live/dead stain) (BioLegend, catalog number: 423102 ), dilution 1:100
Anti CD45- APC-Cy7 antibody (BioLegend, catalog number: 103116 ), dilution 1:500
Anti MHCII- AF700 antibody (BioLegend, catalog number: 107622 ), dilution 1:200
Anti CD3- BV711 antibody (BioLegend, catalog number: 100241 ), dilution 1:500
Anti TCRγδ- PE antibody (BioLegend, catalog number: 118108 ), dilution 1:1,000
Anti CD19- BV650 antibody (BioLegend, catalog number: 115541 ), dilution 1:500
Anti CD49b- PERCP Cy5.5 (BioLegend, catalog number: 108916 ), dilution 1:200
Anti CD11b- BV421 antibody (BioLegend, catalog number: 101235 ), dilution 1:200
Anti Ly6C- APC antibody (BioLegend, catalog number: 128016 ), dilution 1:200
Anti Ly6G- FITC antibody (BioLegend, catalog number: 127606 ), dilution 1:500
Anti F4/80- BV605 antibody (BioLegend, catalog number: 123133 ), dilution 1:200
Anti CD11c- PECy7 antibody (BD Pharmingen, catalog number: 561022 ), dilution 1:500
Anti Cytokeratin-PE antibody (Abcam, catalog number: Ab52460 ), dilution 1:500
Ice
Dimethyl sulfoxide (DMSO)

Equipment

Micropipettes
Tube holders and racks
Ice holder
Biosafety cabinet (BSL2)
Rotator (Fisher Scientific, UVP HB-1000 Hybrodizer Hybridization Oven, catalog number: UVP 95003001 )
Centrifuge (Eppendorf, model: 5810 R )
Rare earth permanent magnet (Promega, Magnesphere^® Technology magnetic separation stand, catalog number: Z5343 )
Incubator 37 °C with 5% CO₂(Thermo Scientific, Heracell 160I, model: 41774412 )
Nexcelom cell counter (Nexcelom Bioscience, Cellometer auto T4 automated cell counter, model: Auto T4-003-0489 )
Flow cytometer (BD Biosciences, model: LSR-II )

Software

BD FACSDiva software (BD Biosciences, CA, USA)
Flowjo software version 10 (BD Biosciences, CA, USA)

Procedure

Preparation of digestion media
1. Prepare digestion media just before use. Fresh media has to be used for each experiment. To DMEM high glucose media, add 10% heat inactivated FBS (HI-FBS), 0.1% collagenase Type 4 and 0.005% hyaluronidase. Both collagenase Type 4 and hyaluronidase are in powder form, weigh and add directly to the media. Mix with gentle agitation until all ingredients dissolve. Aliquot 25 ml to each conical tube for each sample.
Sample preparation
1. Euthanize animals previously injected with iron oxide nanoparticles. Excise tumors and record their weight.
2. Mince completely with a razor blade in a Petri dish.
3. Use a syringe plunger to mash the tumor.
4. Transfer to the conical tube containing 25 ml digestion media (25 ml per 150-200 mg tumor weight).
5. Rotate at 37 °C for 30 min and filter through a 100 µm filter while mashing with a syringe plunger.
6. Centrifuge at 335 x g at 23 °C for 10 min.
7. Wash with 25 ml DMEM/tumor with 10% HI-FBS. Resuspend with a pipette and centrifuge again at 335 x g at 23 °C for 10 min.
8. Discard the supernatant and add 3 ml ACK (Ammonium-Chloride-Potassium) lysing buffer. ACK lysing buffer is used to lyse red blood cells from flow preparation. It is commercially available, but can also be prepared with ammonium chloride, potassium bicarbonate, and sodium EDTA. Resuspend with a pipette and leave at room temperature for 3-5 min.
9. Add DMEM with 10% HI-FBS up to 25 ml and centrifuge at 335 x g at 23 °C for 10 min and discard the supernatant.
Magnetic separation
1. Add 4 ml DMEM with 10% HI-FBS.
2. Resuspend and transfer all 4 ml to labeled flow tubes.
3. Keep all tubes upright on a rare earth permanent magnet and incubate for at least 30 min in 37 °C incubator with 5% CO₂.
4. After magnetic separation, carefully collect supernatant into another tube.
5. Add 4 ml of DMEM with 10% HI-FBS to the magnetically attached fraction and resuspend.
6. Centrifuge all tubes at 335 x g at 23 °C for 10 min and discard supernatant. A schema of the method is given in Figure 2.
  
  Figure 2. Overall scheme of magnet-assisted cell sorting from tumor for flow cytometry
Staining and fixation
1. Wash sedimented cells (pellet) with 2 ml of PBS by resuspending using a pipette.
2. Centrifuge at 335 x g at 23 °C for 10 min.
3. Add adequate amount of PBS (usually 1 ml) to enable cell counting, and count cells after dilution.
4. Transfer 2 million cells from each tube and into fresh tubes.
5. Separately, reserve 1-2 million cells for unstained control, live/dead control and for each FMO tubes (FMO: Fluorescence Minus One) for compensation and gating.
6. Centrifuge at 335 x g at 23 °C for 5 min.
7. Discard the supernatant and add 100 µl live/dead solution (stock in DMSO) (1 µl/100 µl PBS) and incubate at room temperature in dark for 30 min.
8. Centrifuge at 335 x g at 23 °C for 3 min.
9. Wash with 100 µl PBS by resuspending with a pipette and centrifuge at 335 x g at 23 °C for 3 min.
10. Discard the supernatant and add 100 µl Fc blocker (1 µl/100 µl PBS) and incubate on ice for 10 min.
11. Centrifuge at 335 x g at 23 °C for 3 min.
12. Wash with 100 µl PBS with 5% HI-FBS by resuspending with a pipette.
13. Centrifuge at 335 x g at 23 °C for 3 min.
14. Add one drop of single stain beads to new tubes without cells.
15. Prepare antibody cocktails (50 µl/sample) in biosafety cabinet in dark.
16. All dilutions are made in PBS with 5% HI-FBS on ice:
  1. For test samples–add all antibodies in appropriate dilution.
  2. For single stain beads–single antibody in appropriate dilution.
  3. For FMO–all antibodies minus one antibody in appropriate dilution (e.g., if using 12 antibodies, 12 separate antibody cocktails should be prepared each with all 11 antibodies without 1 antibody).
17. Add 50 µl of the antibody cocktail to their respective tubes, and mix gently with pipette and incubate at room temperature in dark for 30 min.
18. Centrifuge at 335 x g at 23 °C for 3 min.
19. Wash with 100 µl PBS with 5% HI-FBS by resuspending using a pipette.
20. Centrifuge at 335 x g at 23 °C for 3 min.
21. Prepare fixation/ permeabilization (fix/perm) solution by mixing 1 part of concentrate with 3 parts of diluent as per manufacture protocol.
22. Resuspend in 50 µl of fix/perm solution and incubate at room temperature in dark for 30 min.
23. Centrifuge at 335 x g at 23 °C for 3 min.
24. Wash with 100 µl PBS with 5% HI-FBS by resuspending using a pipette.
25. Centrifuge at 335 x g at 23 °C for 3 min.
26. Discard the supernatant and add 200 µl PBS with 5% HI-FBS. Run samples in flow cytometer LSR-II (BD Biosciences, San Jose, CA).
27. Can be stored in dark at 4 °C covered with aluminum foil for running next day (within 24 h).
Data Acquisition
1. Acquire data in LSR II using BD FACSDiva software. First, run single stain controls to set up negative and positive gates. Then run FMOs and samples to record data collected from minimum of 200,000 events per sample. Export data as FCS files after compensation in BD FACSDiva software ( Staats et al., 2019 ).

Data analysis

Use Flowjo software for analyzing data.
Export FCS files to Flowjo software and group them as FMOs and Tumor samples.
Open one FMO file and plot SSC-A vs. FSC-A.
Gate on all cells–P1.
Select P1 and plot FSC-H vs FSC-A to select single cells–P2.
Select P2 and plot SSC-A vs Live/dead to exclude dead cells. Gate on negative population–P3
Select P3 and plot Live/dead vs. CD45+ve population. Use CD45 FMO to set up gate. Here in CD45 FMO, the signal for CD45 will be negative. This helps to set up gates in unbiased manner. Likewise, set up gates based on FMOs for all other colors. Use the same set up for all the samples by dragging the all samples gate of final set up to tumor samples group.
Identify cell populations based on the specific markers as given below. Gating strategy is given in Figure 3 and in Figure S8 of original paper ( Korangath et al., 2020 ).
1. Tumor cells–CD45-ve, CK+ve
2. T cells–CD45+ve, CD3+ve
3. Gamma delta T cells–CD45+ve, CD3+ve, TCR+ve
4. B cells–CD45+ve, CD3-ve, CD19+ve
5. NK cells–CD45+ve, CD3-ve, CD49b+ve
6. Neutrophils–CD45+ve, CD3-ve, CD49b-ve, CD19-ve, CD11b+ve, Ly6G+ve
7. Dendritic cells–CD45+ve, CD3-ve, CD49b-ve, CD19-ve, MHC II+ve, CD11c+ve, F4/80-ve
8. Monocytes–CD45+ve, CD3-ve, CD49b-ve, CD19-ve, CD11b+ve, Ly6G-ve, Ly6C+ve, F4/80-ve
9. Tumor Associated Macrophages–CD45+ve, CD3-ve, CD49b-ve, CD19-ve, CD11b+ve, Ly6G-ve, Ly6C-ve, F4/80+ve
*Two separate tubes of same samples were used for tumor cell (Live/dead, CD45, CK antibodies) and immune subpopulation (Live/dead, CD45, CD3, CD49b, CD19, CD11b, Ly6G, Ly6C, F4/80, MHC II, CD11c, TCR antibodies) identification.

Figure 3. Flow cytometry gating strategy to identify different cell population

Acknowledgments

This protocol is derived from a published study (Korangath et al., 2020) funded by the Jayne Koskinas Ted Giovanis Foundation for Health and Policy. In addition, partial support from NIH 5R25GM109441 and NIH/NCI grant P30 CA006973 were used. Authors wish to acknowledge Ms. Tricia L Nilles for assistance in flow cytometry experiments.

Competing interests

R.I. is an inventor on nanoparticle patents. All patents are assigned to either The Johns Hopkins University or Aduro BioTech, Inc. R.I. consults for Imagion Biosystems, a company developing imaging with magnetic iron oxide nanoparticles; and, for Magnetic Insight a company developing magnetic particle imaging technology. All other authors report no conflicts of interest.

Ethics

All animal studies were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University.

References

Hedayati, M., Abubaker-Sharif, B., Khattab, M., Razavi, A., Mohammed, I., Nejad, A., Wabler, M., Zhou, H., Mihalic, J., Gruettner, C., DeWeese, T. and Ivkov, R. (2018). An optimised spectrophotometric assay for convenient and accurate quantitation of intracellular iron from iron oxide nanoparticles. Int J Hyperthermia 34(4): 373-381.
Korangath, P., Barnett, J. D., Sharma, A., Henderson, E. T., Stewart, J., Yu, S. H., Kandala, S. K., Yang, C. T., Caserto, J. S., Hedayati, M., Armstrong, T. D., Jaffee, E., Gruettner, C., Zhou, X. C., Fu, W., Hu, C., Sukumar, S., Simons, B. W. and Ivkov, R. (2020). Nanoparticle interactions with immune cells dominate tumor retention and induce T cell-mediated tumor suppression in models of breast cancer. Sci Adv 6(13): eaay1601.
Marchal, S., El Hor, A., Millard, M., Gillon, V. and Bezdetnaya, L. (2015). Anticancer drug delivery: An update on clinically applied nanotherapeutics. Drugs 75(14): 1601-1611.
Sheen, M. R., Lizotte, P. H., Toraya-Brown, S. and Fiering, S. (2014). Stimulating antitumor immunity with nanoparticles. Wiley Interdiscip Rev Nanomed Nanobiotechnol 6(5): 496-505.
Staats, J., Divekar, A., McCoy, J. P. and Maecker, H. T. (2019). Guidelines for Gating Flow Cytometry Data for Immunological Assays. In: McCoy, Jr J.(Ed.). In. Immunophenotyping. Methods in Molecular Biology vol 2032. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9650-6_5.
Wu, W., Wu, Z., Yu, T., Jiang, C. and Kim, W. S. (2015). Recent progress on magnetic iron oxide nanoparticles: synthesis, surface functional strategies and biomedical applications. Sci Technol Adv Mater 16(2): 023501.
Zanganeh, S., Hutter, G., Spitler, R., Lenkov, O., Mahmoudi, M., Shaw, A., Pajarinen, J. S., Nejadnik, H., Goodman, S., Moseley, M., Coussens, L. M. and Daldrup-Link, H. E. (2016). Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues. Nat Nanotechnol 11(11): 986-994.