Methods and Materials
Procedural Precautions
Every process must be conducted under a sterile hood. Before the hood is used the UV light must be turned on for ten minutes. All materials that enter the sterile hood are sprayed down with a solution of 70% ethanol, including gloves and bottles. All bottles are not opened until completely under the hood. All materials are sterile plasticware. When materials or solutions are not in use they are closed, in an attempt to minimize contamination. Pipette tips were changed every time a new medium was entered.
Culture Growth
Mammalian cells are cultured in flasks that are coated with 2ML of trypsin solution, in a humidified atmosphere, consisting of 5% ofCO2,and maintained at a temperature of 37℃. Then the 4T1 cells are placed into the culture vessel at a starting concentration of 100,000 cells per mL. The cells were resuspended and the solution was then centrifuged at 500 RFC for 5 minutes. The remaining liquid was then discarded and the cell clump was resuspended in 1 mL of media. This solution was then carefully placed and swirled in a flask and placed in the incubator.
The cells were then confirmed to be 100% confluent, meaning the dish was completely full of cells, using a hemocytometer and standard procedure. Next cells must be fixed, using 4% paraformaldehyde (PFA). Next, the cells were permeabilized with soap (allowing for cell entry), specifically with 1% of Triton-X 100, also for 15 minutes. The cells are then incubated and blocked for thirty minutes with a blocking buffer, preventing non-specific receptor binding. The primary CXCR4 antibody was used at a 1:50 dilution. Secondary antibodies for TFF2 at a 1:5000 dilution, Alexa 488 (fluoresces green; targets TFF2) and Alexa 555 (fluoresces red; targets CXCR4). The cell nuclei are then dyed for about ten minutes with Hoechst at a 1:1000 dilution (2.5 micrograms/mL).
Treatment Groups
The procedure was replicated with controls. In the first control, the cells are only treated with the primary antibody and shouldn’t emit any color due to the fact that the phlouraflors are attached to the second antibody. The next control was treated with only the second antibody, which should also show no color since it cannot bind the cells without the primary antibody. These controls were necessary to ensure the antibodies are specific.
Then both the primary and secondary antibody were included in the treatment of the cells. The fluorescent colored antibodies determined whether or not the cancer cells were shown to be using TFF2 signals to communicate to MDSCs.
Every process must be conducted under a sterile hood. Before the hood is used the UV light must be turned on for ten minutes. All materials that enter the sterile hood are sprayed down with a solution of 70% ethanol, including gloves and bottles. All bottles are not opened until completely under the hood. All materials are sterile plasticware. When materials or solutions are not in use they are closed, in an attempt to minimize contamination. Pipette tips were changed every time a new medium was entered.
Culture Growth
Mammalian cells are cultured in flasks that are coated with 2ML of trypsin solution, in a humidified atmosphere, consisting of 5% ofCO2,and maintained at a temperature of 37℃. Then the 4T1 cells are placed into the culture vessel at a starting concentration of 100,000 cells per mL. The cells were resuspended and the solution was then centrifuged at 500 RFC for 5 minutes. The remaining liquid was then discarded and the cell clump was resuspended in 1 mL of media. This solution was then carefully placed and swirled in a flask and placed in the incubator.
The cells were then confirmed to be 100% confluent, meaning the dish was completely full of cells, using a hemocytometer and standard procedure. Next cells must be fixed, using 4% paraformaldehyde (PFA). Next, the cells were permeabilized with soap (allowing for cell entry), specifically with 1% of Triton-X 100, also for 15 minutes. The cells are then incubated and blocked for thirty minutes with a blocking buffer, preventing non-specific receptor binding. The primary CXCR4 antibody was used at a 1:50 dilution. Secondary antibodies for TFF2 at a 1:5000 dilution, Alexa 488 (fluoresces green; targets TFF2) and Alexa 555 (fluoresces red; targets CXCR4). The cell nuclei are then dyed for about ten minutes with Hoechst at a 1:1000 dilution (2.5 micrograms/mL).
Treatment Groups
The procedure was replicated with controls. In the first control, the cells are only treated with the primary antibody and shouldn’t emit any color due to the fact that the phlouraflors are attached to the second antibody. The next control was treated with only the second antibody, which should also show no color since it cannot bind the cells without the primary antibody. These controls were necessary to ensure the antibodies are specific.
Then both the primary and secondary antibody were included in the treatment of the cells. The fluorescent colored antibodies determined whether or not the cancer cells were shown to be using TFF2 signals to communicate to MDSCs.