Cell linesThis research was conducted on balb/c mice. This was done in order to keep the bloodlines all the same. When starting any cell cultures, the confluency must be up to standard. For this research, the confluency had to be more than 80% and as close to 100%. The media used was RPMI media.
Procedural CautionsFor this research, it was very important for the workspace to be sterile. For this reason, a sterile hood was used, specifically a Laminar Flow Hood. This hood uses U.V. radiation to sterilize the hood before use. Standard procedure is to turn on the U.V. light on for 10 minutes before use. The hood has a glass cover that needs to be closed during this time. Before the items of study were taken into the hood they were sprayed down with a 70% ethanol solution. This was done every time anything is being put into the hood and is done to decrease the chances of contamination. This also includes hands, which are gloved at all times. The flasks are kept closed until safely inside the hood. In the hood, when working with the item of examination, the standard was to change micropipette tips each time (will come back to this).
Making Cell Cultures (4T1 Cancer Cells)In this research, the standard procedure for creating a cell culture was used. The old cell culture was taken into the hood (Laminar Flow Hood). In the hood, the media was removed with a 10 mL surgical pipet. A surgical pipet is battery powered and is usually used for less accurate measurements (Fig 5). After the media was removed, a solution called trypsin was added. This solution is made to stop cells from adhering to the flask wall. 2mL of Trypsin was added and was evenly distributed onto the walls of the flask, making sure the neck of the flask didn’t get contaminated. This was very important to prevent bacterial growth. A micropipette was used for this measurement. A micropipette is used for very exact measurements and are highly tuned installments. The flask was then taken to an incubator so that the trypsin could do its job and detach the cells from the walls. The flask was left at 37° C for about 3-4 minutes or until the cells were off the walls. The cells were then re-suspended in 5 mL of fresh culture media. This was done as to stop the trypsin from killing the cells, for the media has proteins that inactivate trypsin. The cells and media were then transferred into a 15mL conical tube. Once transferred, the micropipette was used to gently mix the solution. This was done to make sure all clumps of cells were dissociated. The tube was then immediately centrifuged as to not let any cells settle. The tube was centrifuged for 5 minutes at 500 RCF (Relative Centrifugal Force). When this was happening the new flask was being prepared. 6mL of fresh media was added to the new flask and the flask was labeled. The centrifuge finished shortly after, and the tube was taken out and brought back to the hood. The supernatant (liquid) was then removed. The cells were then again resuspended in 1 mL of media. Of the resuspended cells, 1 uL was taken and added to the new, labeled flasks. The flasks were then swirled in a figure 8 pattern as to slowly and evenly distribute the media over the cells.
Cell FixationTo look at the 4T1 cancer cells while they are dyed, the process of fixing must first take place. This is when there is a buffer added, in this case, 4% paraformaldehyde (PFA). This fixation buffer freezes the cells in real-time, meaning the cells are dead, but can be observed in the exact same state as of the buffer being added. To start fixation of the cells, 4% PFA was added to the 4T1 cells for fifteen minutes. The cell membranes then needed to be permeabilized. For this, a solution called Triton x 100 (1%) was used for fifteen minutes. CXCR4 receptors on the membrane needed to be blocked. The blocking buffer, two protein-rich solutions (Bovine Serum Albumin and FC Block) that fill out the receptors, was then added. This made it nearly impossible for the CXCR4 to bind anything else. The solutions are left with the cells for approximately 30 minutes to make sure that CXCR4 is blocked. The primary antibodies were then added. One antibody targeted CXCR4 and was there to make sure that CXCR4 wasn’t binding to anything. At the same time, the other primary antibody was added. This antibody targeted TFF2, it is the control antibody. Then the secondary antibodies were added. The one for CXCR4 was Alexa 555 (red). The one for TFF2 was Alexa 488 (green). The numbers that are given after Alexa, are the wavelengths of the antibodies. For the 4T1s, a dye was used to highlight the nucleus. The dye was called Hoechst (blue). The dye is left with the 4T1 for ten minutes. The primary antibodies were at a 1:50 dilution and the secondary antibodies were at a 1:500 dilution. The dye was at a 1:1000 dilution.
Confocal MicroscopyWith all these antibodies and dyes, there must be a way to observe the colors. This is where confocal Microscopy comes in. The confocal microscope uses light to observe the different colors of the specimens. The specimens in this situation were not only easy to view, but also easy to manipulate. This was done though the movement of the plate that the specimens were on. The confocal microscope has a viewing plate that can be moved in very small measurements, allowing the specimen to be moved very carefully. It is so closely calibrated that nothing can touch the table. The confocal microscope is a very fragile piece of scientific equipment but is able to look at things with incredible magnification.
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