Materials and Methods
When conducting the experiment, six BALB/c mice were used. All mice resided in individual cages in a temperature controlled room for the course of this research. The mice were assigned at random to two groups: exercise tumor and sedentary tumor . The exercise group consisted of three mice which were given access to a cage mounted running wheel (11.5 cm in diameter). The three mice had access to this wheel twenty-four hours a day for three weeks before the tumor cells were injected, and had access to this wheel for another twelve days after the injection. The sedentary group was restricted to normal cage activity before and after the injection of the cancer cells. Wheel running distances were monitored using the Vital View data acquisition system (MiniMitter; Bend, OR). Running distances were calculated as (3.14 11.5 cm number of revolutions)/(100 cm/m 1000 m/km).
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Within the mammary gland, orthotopic tumors grew using syngeneic 4T1 mouse mammary carcinoma cells (ATCC, Manassas, VA). The 4T1 cell line was chosen because of previous work showing that these tumors increase the amount of MDSC in the spleens of tumor bearing animals significantly. 4T1 cells were grown in RPMI medium, the RPMI medium was supplemented with 10% fetal bovine serum (FBS) and maintained in an incubator at 37°C. After three weeks of the study, the tumor bearing mice were sedated with isoflurane and injected with 1x104 4T1 cells in the fourth mammary gland. Body weight and body condition were monitored three times per week for the duration of the study.
Tissue Preparation and Flow Cytometric Analysis
On the fifth week of the study, all animals were euthanized with a lethal injection consisting of heparinized Euthasol. Samples of the spleen, blood, and tumor were collected and prepared for analysis using flow cytometry. The spleens were removed from the animal using the frosted end of two microscope slides and filtered through a 100 µm cell strainer then weighed. The blood samples were collected by cardiac puncture into a syringe that contained 0.1 mL of heparin. The tumors were removed, weighed and homogenized using a cell dissociation sieve tissue grinder (Sigma Aldrich). The next step was to enzymatically digest the sample in type I collagenase (1 mg/mL) for about 45 minutes at 37°C on a platform rocker. After the digestion, the tumor samples were then filtered through a 100 µm cell strainer. Single cell suspension of splenocytes, tumor, and surrounding blood were stripped of red blood cells by being incubated in buffer, ammonium chloride potassium lysis, for 3 minutes at room temperature.
MDSC Sorting Using Fluorescence Activated Cell Sorting
PMN-MDSC and M-MDSC were identified from the spleens of the tumor bearing mice for assessing using fluorescence activated cell sorting (FACS). Gates were established to identify CD11b+Ly6G+Ly6Clo and CD11b+Ly6G-Ly6Ch cells for sorting. The subpopulations of MDSC were grouped together and sorted into the same 15 mL container that contained RPMI (cRPMI) supplemented with 50% PBS using a SH800S cell sorter. After sorting, cells were centrifuged for 7 minutes at 300g. The cell pellet was resuspended in cRPMI growth medium at a concentration of 1x106/mL and set aside.