Image captured on The EVOSĀ® FL Auto Imaging System by the author
It has been a very long time since I last posted on HIVE. I hope everyone is doing well and life is being kind to all of you. I took a much needed break from the internet. I am here today to give an update of where I am in my research these days. For those new to my blog, I am a laboratory technician doing human clinical research to better understand the immunological mechanisms of sepsis and why sepsis patients and the healthy aging population are more susceptible to fungal infections, especially Candida albicans.
I have been working with HL-60 cells for quite some time now, but finally came to the conclusion that genetic manipulation of this cell type was detrimental and probably caused the cells to differentiate and die. I could not keep them alive long enough to conduct any quality experiments. At least I was able to show the transfection worked, as you can see in the image above. The image clearly shows the anticipated red fluorescent protein tag. The blue stain is Dapi to show the nucleus of live cells. You can see that most of the transfected cells are dead. Purple indicates live transfected cells.
I decided at the end of last year to switch to a new cell type, K-562. This is a Chronic Myeloid Leukemia cell line. HL-60 cells are an Acute Promyelocytic Leukemia cell line. In my work with HL-60s, I discovered these cells become activated and differentiate into neutrophil like cells under stressful conditions, such as overgrowth or transfection protocols. This lead me to find a new cell type to perform the transfection.
For several weeks now, I have been familiarizing myself with this new cell type. They grow much faster than HL-60s, so I must passage them a little differently at a much lower cell concentration. They do not seem to be affected by overgrowth, so that is a plus! I just recently transfected this cell type, so it's a little early to tell if the transfection is successful and viable. It may take a few more weeks for the transfected cells to proliferate. However, I did confirm yesterday on the fluorescent microscope that the red tag is present in these cells. Now, I wait for them to grow.
We discovered in our research that sepsis patients have an increased amount of a certain protein on the cell surface of their neutrophils. Leukemia cells are neutrophil-like cells, which is why I chose HL-60 cells in the first place. My hope is to overexpress this protein found on sepsis patient cells on the leukemia cells. Like a model sepsis cell. Once I have created a stable transfected cell, we can conduct the same experiments we did on the sepsis patient's, as well as healthy donor cells. We will be able to compare the results with a cell that has this protein overexpressed. What will this tell us? It may shed some light as to why this protein is upregulated on sepsis patient cells compared to healthy cells. It could be a detrimental overexpression due to infection or it could be a protective mechanism.
I will write again as soon as I have more images and my newly transfected cells actually grow! Thanks for reading and for your support! Have a great day!
