-
Micropipette tips (Rainin, catalog numbers: 17005860 [20 µl]; 30389240 [200 µl]; 30389213 [1,000 µl])
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Corning® non-treated culture dishes (D × H 100 mm × 20 mm) (Corning, catalog number: 430591)
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Syringe (3 ml) (Fisher Scientific, catalog number: 14-823-436)
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Conical tube (50 ml) (Thermo Scientific, catalog number: 339653)
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Cell strainer Nylon (100 µm) (BD Falcon, catalog number: 352360)
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Aluminum foil
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IntegraTM MiltexTM Sterile Surgical Blades (Fisher Scientific, catalog number: 12-460-444)
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Microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-137)
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FalconTM Round-Bottom Polystyrene Tubes (Flow tubes) (Corning, catalog number: 352054)
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ACK lysing buffer (Quality Biologicals, catalog number: 118-156-101)
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Dulbecco’s Modified Essential Medium (DMEM) high glucose (MilliporeSigma, catalog number: D5796)
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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: 02151272)
-
Phosphate buffered saline (PBS) (Corning Cellgro, catalog number: 21-040-CV)
-
OneComp eBeads (eBioscience, catalog number: 01-1111-42)
-
eBioscienceTM Fixation/Permeabilization Concentrate (Thermo Scientific, catalog number: 00-5123-43)
-
eBioscienceTM Fixation/Permeabilization Diluent (Thermo Scientific, catalog number: 00-5223-56)
-
Anti CD16/32 antibody (Fc blocker) (BioLegend, catalog number: 101302), dilution 1:100
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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)
-
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
).
-
Tumor cells–CD45-ve, CK+ve
-
T cells–CD45+ve, CD3+ve
-
Gamma delta T cells–CD45+ve, CD3+ve, TCR+ve
-
B cells–CD45+ve, CD3-ve, CD19+ve
-
NK cells–CD45+ve, CD3-ve, CD49b+ve
-
Neutrophils–CD45+ve, CD3-ve, CD49b-ve, CD19-ve, CD11b+ve, Ly6G+ve
-
Dendritic cells–CD45+ve, CD3-ve, CD49b-ve, CD19-ve, MHC II+ve, CD11c+ve, F4/80-ve
-
Monocytes–CD45+ve, CD3-ve, CD49b-ve, CD19-ve, CD11b+ve, Ly6G-ve, Ly6C+ve, F4/80-ve
-
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
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.
All animal studies were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University.