I began to research TNF and HIV in an effort to comprehend my experiment and data. So far, I found two hypotheses that I need to look into: TNF activates the NF-kappa B pathway, activating the NF-kB receptors which HIV then use to infect the cell; HIV attaches to and infects cell via TNF receptors.
I made some more devices for Laura. The lab got two new master slides for the flow-patterning devices and Laura did not really like them. She said that they were too sticky. I found that they were the same, mainly because I did not really care. I found that the area in which I poured the PDMS produced the most bubbles, so I focused on pouring PDMS in empty spaces so I could pop less bubbles. I also punched holes for the inlets and outlets for some flow-patterning devices for Laura. It took approximately 20-30 minutes (I used music as a method to keep track of time).
I did some more research on TNF, but I also asked Victor about why TNF activated HIV. He said that the TNF pathway activates NF-kappa B, AP1, and SP1 receptors, as well as the NF-kappa B pathway and the HIV uses that pathway to infect the cell and proliferate. However, I also found a paper from France that explains how HIV actually takes over the TNF pathway. Victor asked me to send him that paper, so I did. I need to read that French paper, but I also need to learn how HIV takes over the NF-kappa B pathway.
I also ran the TNF experiment again. However, this time I changed the amount of media in the devices at the end since Victor believes that we lost the cells because flow was insufficient. I ran the same procedure: I plasma bonded the devices, I put DI water in the devices to ensure flow can occur, I primed the devices with RPMI, loaded my cells, washed the cells with RPMI, stained them with Hoechst, then loaded the control devices with RPMI and the manipulated devices with TNF. I left them to incubate with a humidifier for 24 hours. The trapping efficiency in the devices were not too great, but most of them were above 80%, so it should be fine.
Victor did not show up today, and will not show up for the rest of the week. He said he can come in tomorrow and prepare cells for another experiment that I can run on my own. I will have to prepare the cells properly and without assistance (Ay, so much pressure, hopefully I don't break anything).
I learned that HIV cannot use TNF pathway to infect cells, only the NF-kappa B pathway. I began to read a paper on how HIV infects the NF-kappa B pathway in an effort to better understand what is going on inside the devices.
Victor forgot to put me in the schedule for the microscope...
Someone else scheduled the microscope for the whole day (11-6). Now, I have to use another microscope, the Nikon. I need to remember to put myself on the schedule for the EVOS microscope when I do the experiment again tomorrow. This could've been really bad. Thank god for Laura, saving my butt again.
Speaking of Laura, I cleaned some of her devices and punched holes in 6 of them now. It used to be 4, but since we got the new master slide for the flow-patterning device, we have 6. Hooray. More work. (punching holes in those devices take forever, like 30-45 minutes. Thank god for music.)
Victor did not come again today. He said he has a family emergency. Laura will be preparing my cells, but instead of stimulating them with TNF, we will be stimulating them with LPS.
LPS (lipopolysaccharides) are large molecules found in the outer membrane of gram-negative bacteria (bacteria that don't have much peptidoglycan--substance that forms the cell wall of bacteria). It acts as an endotoxin and induces an immune response in humans when it binds to receptors, such as the CD14 receptor. Downstream, LPS activates NF-kappa B via CD14 pathway, therefore it is likely that LPS will activate HIV in the cells.
While doing experimentation, I was supposed to make a solution of LPS and RPMI so the cells can be stimulated by LPS while living in a favorable environment. However, I accidentally put in pure LPS into two devices and killed the cells. Laura helped me get more LPS, and I made the solution for the devices. I had to take out everything from the two devices I accidentally messed up. The devices were still intact and undamaged, so that was good. I redid the procedure for the two devices while the rest of the devices were still fine. I constantly checked on the devices under each microscope to ensure that cells were still trapped and the device was functioning properly. I then put a humidifier into the container with the devices at 1:25 pm, so by 1:25 pm tomorrow, I will need to image my cells.
Also, I realized that no one taught me the cleaning procedure for experiments. I asked Laura, and she taught me what to toss out, what I can immediately throw in the trash and what I have to bleach first, and how to sterilize the workbench properly.
Most of the cells in my devices disappeared... once again. After looking at my devices, I realized why the cells were disappearing. The flow pressure from the large amount of fluid in the inlet caused cells to escape the traps and end up in the outlet. I would like to try decreasing the amount of liquid in the inlets and outlets, but I do not want to put in so little that it could equilibrate so easily, which would disrupt flow. Laura did not prepare the Hoechst stain properly, so I could not see the cells under the DAPI channel (filter to see stain). I imaged what I could, but it is likely that I will not even bother to gather data from these images. I did notice that LPS slightly activated HIV in some cells, while inducing apoptosis in others.