Today, I did some cell culturing and split my cell groups in a ratio of 1:5 (1 mL of 1 mil cells:5 mL of entire solution (cells and RPMI medium)). This was the 29th passage, or the 29th time these cells have been split. I took out 7.5 mL of cells from J65C 6.6 and left 2 mL in container to grow. I placed the 7.5 mL of cells into a 15 mL falcon tube to spin in the centrifuge to get a pellet of cells at the bottom of the tube to run through three devices. Once I got a pellet, I removed most of the old medium and added 500 microliters of new RPMI into the tube. I then took the entire solution out of the falcon tube and into two cell strainers to ensure the cells were separate and not in clumps. I ran them through 3 devices I prepared earlier. After a few minutes, I removed the leftover media and replaced it with Hoechst, a fluorescent staining agent that stains the DNA in the nucleus of cells. I then placed the devices in a styrofoam box to block off all of the light since the Hoechst is light-sensitive. After 20+ mins, I removed the Hoechst and replaced it with media so the cells can survive. I then checked the devices under the microscope to ensure that cells were trapped and no more cells were flowing, otherwise it would interfere with the imaging process. After, Victor brought me to the EVOS microscope, a widefield fluorescence microscope used for cell imaging. Victor showed me how to use the device, and I took pictures of the cells trapped in the channels of the devices. I used multiple filters to tell us different things about the cells. For example, the GFP filter shows if there is HIV present in cells by activating the GFP reporter attached to the end of HIV strains and making the cells appear green. The TxRed filter shows if there are proteins that stimulate the production of HIV and make the cells orange. The DAPI filter makes the cells' nuclei appear blue since it reveals the Hoechst stain. I took the images and used ImageJ to observe the pictures on my computer.
Uneventful day. I did not do anything new, just made some more devices for Laura so she can do her experiment. I did make two masters (not the device themselves, just casts) for future use. I also punched out holes in four flow-patterning devices for Laura using a drill press in a lab on the first floor. It took approximately 30 minutes to complete. For the procedure on how I made the devices, refer to Lab Internship: Week 2.
I also read articles on cell signaling, which is what Laura is focusing her project on.
Laura informed me that she and Victor will not be in tomorrow, but I will still come in so I can pass some cells (cell culturing). After that, I plan on reading for a while before heading home.
I split J65C 4.4 and 6.6 1:5 successfully on my own. I did not die, nor did the cells or station catch on fire, so I think it was an overall success, unless I missed something that I did not realize. I warmed up my media, I made sure everything was clean and sterile, I did not spill the cells or kill them on accident, I did not break anything, I pipetted the solutions multiple times to get rid of clumps of cells, I only made a single bubble (which is pretty good), and I put in the proper amount of media (8 mL to go with the 2 mL of cells to make 10 mL of solution). The solutions should be at 200,000 cells per mL now. If I messed something up, Victor will notice when he splits the cells on Saturday.
Laura is not here, she is actually on vacation. I do not have much else to do besides do some readings and extra work that I have.