To determine the effects of exercise on MDSCs and T cells, A 4T1 line of breast cancer was injected into the mammary glands of female BALB/c mice. The 4T1 tumor model is representative of an aggressive and late stage breast cancer and closely models the progression of human breast cancer. The one injected mouse was put into an individual cage with a wheel for it to run on voluntarily. The other cancerous mouse was put into a cage without wheels and therefore did not exercise. Another control mouse was not injected with cancer and did not exercise. Daily measurements of the size of the tumors were taken on the mice that were injected with the 4T1 cells to examine the growth and progression of cancer. After four weeks the mice were euthanized in order to perform various assays to test the predicted hypothesis.
Harvesting Mouse Peripheral T Cells and MDSCs
In order to collect T cells, the mouse was euthanized by a heparinized sodium pentobarbital overdose. The spleen was then removed and placed in Complete RPMI. Then using the frosted end of 2 microscope slides the spleen was dissected and filtered through using a 70µm strainer. After centrifuging the sample for 5 to 10 minutes, the cells were resuspended in a 3ml ACK lysis buffer and then incubated at room temperature for 3 minutes. After, it was necessary to quench the lysis buffer with 6 mL of complete RPMI and again centrifuge for 5 to 10 minutes. The next step was to label the cells with CD8. First, the cells were resuspended in 300µL of flow incubation buffer and 100µL of cell solution was transferred to a 1.5mL Eppendorf tube. In the Eppendorf tube 2µL of Fc block was added and then the tube was incubated on ice for 5 minutes. After incubating, 700µL of flow buffer was added and the tube was centrifuged for 5 mins. The cells were then resuspended in CD8 antibody solution and then incubated on ice in the dark for 45 minutes. Then the process of sorting began. First, the cells were washed by adding 700µL of flow buffer and then centrifuged at 4℃ for 5 mins. This step was repeated again and then the cells were resuspended in 300-700µL flow buffer. This amount was dependant on the cell concentration, if the cell concentration was lower less volume was required. Then using the Sony cell sorter, 1x10^6 CD8 cells per animal were collected in a 15mL conical that contains 3 mL of complete RPMI. Once completed, the T cells were to be stored in a 37℃ incubator overnight to let the cells rest. In order to harvest the mouse MDSC cells the same procedure was followed, except when identifying MDSCs theses cells were labeled an antibody for CD11b, Ly6C, and Ly6G and also when using the Sony cell sorter 2x10^4 MDSC/animal cells must be collected.
T Cell Suppression Assay
The T cell suppression assay was used to measure the ability of T cells to proliferate or grow and divide. The materials necessary for this assay include: a sterile PBS, anti-mouse CD3e, anti-mouse CD28, complete RPMI, CD8 T cells and MDSCs, and 96-well flat-bottom microtiter plates with lids. First, the plate microwells were coated with antibody. Fifty µL of 10 µg/mL anti-CD3e per well. In the unsimulated well 50 µL of sterile PBS was added. Then, the plates were covered with parafilm and incubated at 37℃ for 2 hours. After, the antibody solution was removed with a multichannel pipette and rinsed with 200µL of sterile PBS. This step was repeated again in order to remove unbound antibody. The next step was labeling the T cells with CFSE. To begin this process the T cells were resuspended in 1 mL of RPMI medium and the tube was laid horizontally. Next, 110 µL of PBS was added to the top of the tube to prevent the cell solution from contacting the PBS. After 1.1µL of 5mM stock was added to the PBS. The tube was then capped, quickly inverted and then vortexed for 10 to 15 seconds. Next, the sample was incubated for five minutes at room temperature. It was important to note that now the sample was fluorescent and prone to bleaching and therefore should be kept in the dark. Next, the cells were washed with 10 mL of PBS supplemented with 5% FBS and centrifuged for five minutes. The supernatant from this step should be discarded and then the whole process should be repeated. Once completed, the sample should be resuspended in complete RPM1. After, it was necessary to calculate cell concentration needed for a 4:1 T cell to MDSC ratio. 100 µL of T cell suspension should be added to each well and 100 µL of MDSC suspension in separate wells. 2µg/mL of soluble anti-CD28 should be added to each well and all the wells should be incubated for 4 days. The resulting CFSE dilution peaks should be analyzed using flow cytometry.
Harvesting Mouse Peripheral T Cells and MDSCs
In order to collect T cells, the mouse was euthanized by a heparinized sodium pentobarbital overdose. The spleen was then removed and placed in Complete RPMI. Then using the frosted end of 2 microscope slides the spleen was dissected and filtered through using a 70µm strainer. After centrifuging the sample for 5 to 10 minutes, the cells were resuspended in a 3ml ACK lysis buffer and then incubated at room temperature for 3 minutes. After, it was necessary to quench the lysis buffer with 6 mL of complete RPMI and again centrifuge for 5 to 10 minutes. The next step was to label the cells with CD8. First, the cells were resuspended in 300µL of flow incubation buffer and 100µL of cell solution was transferred to a 1.5mL Eppendorf tube. In the Eppendorf tube 2µL of Fc block was added and then the tube was incubated on ice for 5 minutes. After incubating, 700µL of flow buffer was added and the tube was centrifuged for 5 mins. The cells were then resuspended in CD8 antibody solution and then incubated on ice in the dark for 45 minutes. Then the process of sorting began. First, the cells were washed by adding 700µL of flow buffer and then centrifuged at 4℃ for 5 mins. This step was repeated again and then the cells were resuspended in 300-700µL flow buffer. This amount was dependant on the cell concentration, if the cell concentration was lower less volume was required. Then using the Sony cell sorter, 1x10^6 CD8 cells per animal were collected in a 15mL conical that contains 3 mL of complete RPMI. Once completed, the T cells were to be stored in a 37℃ incubator overnight to let the cells rest. In order to harvest the mouse MDSC cells the same procedure was followed, except when identifying MDSCs theses cells were labeled an antibody for CD11b, Ly6C, and Ly6G and also when using the Sony cell sorter 2x10^4 MDSC/animal cells must be collected.
T Cell Suppression Assay
The T cell suppression assay was used to measure the ability of T cells to proliferate or grow and divide. The materials necessary for this assay include: a sterile PBS, anti-mouse CD3e, anti-mouse CD28, complete RPMI, CD8 T cells and MDSCs, and 96-well flat-bottom microtiter plates with lids. First, the plate microwells were coated with antibody. Fifty µL of 10 µg/mL anti-CD3e per well. In the unsimulated well 50 µL of sterile PBS was added. Then, the plates were covered with parafilm and incubated at 37℃ for 2 hours. After, the antibody solution was removed with a multichannel pipette and rinsed with 200µL of sterile PBS. This step was repeated again in order to remove unbound antibody. The next step was labeling the T cells with CFSE. To begin this process the T cells were resuspended in 1 mL of RPMI medium and the tube was laid horizontally. Next, 110 µL of PBS was added to the top of the tube to prevent the cell solution from contacting the PBS. After 1.1µL of 5mM stock was added to the PBS. The tube was then capped, quickly inverted and then vortexed for 10 to 15 seconds. Next, the sample was incubated for five minutes at room temperature. It was important to note that now the sample was fluorescent and prone to bleaching and therefore should be kept in the dark. Next, the cells were washed with 10 mL of PBS supplemented with 5% FBS and centrifuged for five minutes. The supernatant from this step should be discarded and then the whole process should be repeated. Once completed, the sample should be resuspended in complete RPM1. After, it was necessary to calculate cell concentration needed for a 4:1 T cell to MDSC ratio. 100 µL of T cell suspension should be added to each well and 100 µL of MDSC suspension in separate wells. 2µg/mL of soluble anti-CD28 should be added to each well and all the wells should be incubated for 4 days. The resulting CFSE dilution peaks should be analyzed using flow cytometry.