Flow cytometry
Tumor, blood, and tumor draining lymph nodes were harvested and processed for flow cytometric analysis. 4–5 animals were used per group. Tumor tissue was minced and incubated in Collagenase III (Worthington) for 30 min at 37 °C. After incubation, tissue was passed through a 70 μm nylon cell strainer to produce a single cell suspension. Draining LNs were identified at time of dissection as the most enlarged node adjacent to the tumor. After harvesting, these were similarly processed by mechanical separation into single cell suspension. After centrifugation, red blood cells were lysed using RBC lysis buffer (Invitrogen), using HBSS to neutralize the lysis buffer. Bone marrow was harvested using the femur and tibia. These bones were placed into small 0.6 mL microtubes with a hole punctured in the bottom using an 18-gage needle. These were then stacked into 1.5 mL microtubes and spun at 10,000 rpm for 5 min. Bone marrow was harvested from pellets that had collected in the bottom of the larger microtube.
Cells were transferred into 24 well plates and incubated with monensin and brefeldin to prevent release of cytokines and stimulated with PMA/ionomycin cocktail for 4 h at 37 °C. Following incubation, cells were incubated in FC block (CD16/CD32 antibody, Tonbo bioscience) for 15 min at 4 °C. Cells were then incubated in Live/Dead Fixable Aqua Viability Stain Kit (Invitrogen) in the dark for 20 min at 4 °C. Cells were then stained for surface markers and incubated for 20 min at 4 °C. For analysis of immune cells, the following antibodies were used:
PerCP–CD45 (clone: 30-F11, Biolegend), eF450–CD3 (clone: 17A2, Invitrogen), BUV496–CD4 (clone: GK1.5, BD Biosciences), APC-eF780–CD8 (clone: 53–6.7, Invitrogen), BV570-CD44 (clone: IM7, Biolegend), BUV750–CD25 (clone: PC61, Biolegend), BV785–CD62L (clone: MEL-14, Biolegend), APC-Fire810–PD1 (clone: 29F.1A12, Biolegend), BUV615–CD11c (clone: N418, Invitrogen), PE/Dazzle 594–CCR7 (clone 4B12, Biolegend), BUV661–CD11b (clone: M1/70, BD Biosciences), BUV563–CD19 (clone: 1D3, Invitrogen), PE-Cy5–Ki67 (clone: SolA15, Invitrogen), AF532–FoxP3 (clone: FjK-16s, Invitrogen), BV605–IL2 (clone: MQ1-17H12, BD Bioscience), BUV737–IFNγ (clone: XMG1.2, BD Biosciences), APC–IL10 (clone: 554,468, BD Biosciences), BV421–Tbet (clone: 4B10, Biolegend), PE-Cy7–GRMB (clone NGZB, Invitrogen),
PerCP–Cy5.5-KLRG1 (clone: 2F1, BD Biosciences), BV711–CCR8 (clone: SA214G2, Biolegend), AF647–CD127 (clone: A7R34, Biolegend), AF488–TCF1 (clone: IC8224G, R&D Systems), PE–TOX (clone: TXRX10, Invitrogen), BV650–CXCR3 (clone: CXCR3-173, Biolegend), BUV395–CD103 (clone: 2E7, BD Biosciences), AF700–MCHII (clone: M5/114.15.2, Biolegend).
For analysis of bone marrow, the following antibodies were used:
Lin-FITC (clone: 145-2C11, RB6-8C5, RA3-6B2, Ter-119, M1/70, Biolegend), Sca1-PerCP (clone: D7, Biolegend), cKit-APC (clone: 2B8, Invitrogen), CD34-PeCy7 (clone: HM34, Biolegend), CD48-APC-Fire750 (clone: HM48-1, Biolegend), CD135-BV421 (clone: A2F10.1, Invitrogen), CD150-PE (clone: 9D1, Invitrogen), CD25-BV711 (clone: PC61, Biolegend), CD44-BV570 (clone: IM7, Biolegend), B220-BV750 (clone: RA3-6B2, Biolegend).
After surface staining, cells were fixed and permeabilized using the FoxP3 perm/fix kit (Invitrogen) overnight. Following incubation, cells were stained for intracellular markers and incubated for 30 min at 4 °C. Samples were then run on a Cytek Aurora spectral cytometer at the University of Colorado Diabetes Research Center Flow Cytometry Core. Fluorescence minus one (FMO) controls were used to determine gating strategy. Flowjo (Ashland, OR) software was used for gating and data analysis. Percentages of cell subtypes, such as CD4+ and CD8+ , were used to account for differences in tumor size.