Killing Leukemia Cells In vitro
I started an antibiotic kill curve for the HL-60 cells last week. I need to determine the optimal antibiotic concentration for selection of transfected cells with an antibiotic resistance gene. My hope is to overexpress a certain protein of interest with a fluorescent tag on the cell surface. My main goal is to create a model sepsis cell.
I have been using 200 ug/mL of Hygromycin B antibiotic, a growth inhibitor of mammalian cells, to select for transfected HL-60s in my preliminary studies. The transfection is performed using a plasmid with a CMV promoter containing the genetic code for our protein of interest, an OFP Spark red fluorescent protein and the Hygromycin B resistance gene.
I wanted to pinpoint the concentration needed to select my transfected HL-60 cells in order to develop a stable cell line. I want to compare the immunological responses of these transfected cells to healthy and septic neutrophils.
HL-60 cells under a light microscope, captured by cellphone
Why are HL-60s Important?
Human Leukemia Cell Line 60 was originally isolated from the peripheral blood of a young female in the late 1970s. This patient suffered from acute promyelocytic leukemia. These cancerous cells originated from the overgrowth of immature white blood cells in the bone marrow. You can chemically induce them to be neutrophil-like. I'm planning to use All-Trans Retinoic Acid
This cell line has been used in numerous research studies over the years. HL-60s were one of the first leukemia cells to be cultured in a continuous suspension and thoroughly characterized in the lab. They have been used for cell biology studies including the following:
- cell formation
- cell differentiation
- apoptosis (aka programmed cell death)
- cancer research
I've been working with this cell line since last year. We were informed during a meeting that we needed to prove that our protein of interest has either a detrimental or protective immunological effect when overexpressed. We discovered during our research of healthy donors and sepsis patients that this particular protein is upregulated on the septic neutrophil.
We were also advised to collect patient blood from the infectious disease clinic. We needed to be certain that this upregulation was only occurring in sepsis patients. Interestingly enough, the infectious disease patients have all shown a lower expression of this protein on their neutrophils than healthy donors. We only need one more of these patients for statistical analysis. Healthy donors and sepsis patients will be ongoing for the duration of the study.
What are Neutrophils?
"Neutrophils are a type of white blood cell (leukocytes) that act as your immune system’s first line of defense." - Neutrophils - Cleveland Clinic
We isolate neutrophils from whole blood using immunomagnetic negative selection with a kit from STEMCELL Technologies Inc. Several experiments are performed with these isolated cells to determine the levels of different pattern recognition receptors, uptake efficiency of fungal particles and reactive oxygen species production. These tasks are performed using the BD LSRFortessa X-20 Cell Analyzer from BD Biosciences. I will repeat all of the same experiments with the stable transfected cell line.
I also test cytokine production from isolated cells by Enzyme Linked Immunosorbent (ELISA) and Electrochemiluminescent (ECL) assays. I have to go in late at night when we get samples to collect their growth media and freeze at -80°C. This is to test for levels of these chemical messages that neutrophils use to call on other immune cells.
BD LSRFortessa X-20 Cell Analyzer
Sepsis or Septic Shock
Sepsis is a leading cause of mortality in hospitals. The World Health Organization reported in September 2020 on the "global epidemiology and burden of sepsis, estimating that the life-threatening reaction to infection causes 1 in 5 deaths worldwide." - WHO says sepsis causes 20% of global deaths
Sepsis can lead to septic shock which is marked by a drastic plummet in blood pressure. The risk of death is increased significantly if sepsis progresses to septic shock. The ICU patient has a high death rate at an alarming 40% mortality. Even if they do recover, the immune system is never the same. What causes this massive rate of death?
Neutrophils get angry.
Neutrophils are one of the first immune cells to show up to the site of infection. They are like the soldiers of the immune system that call in the heavy artillery used in an attack. Neutrophils are extremely sensitive immune cells. They get stimulated or activated very easily.
When there is bacteria or fungus in the bloodstream, as in sepsis, the reaction can be catastrophic. Neutrophils will explode to release cytotoxic substances to kill invaders. They cause a flood of cytokines that create an overabundance of inflammatory reactions to destroy whatever organism has made them mad. This cytokine storm can be deadly to the sepsis patient, damaging tissues and organs in it's wake.
Experimental Design
In order to pinpoint the optimal concentration of antibiotic, I needed several doses of Hygromycin B. The stock solution is 50 mg/mL which needs to be diluted to a working solution of 5 mg/mL. This working solution is then added to a glucose rich growth media. Cancer cells love sugar!
I did go a little overboard and made 10 different antibiotic concentrations. I have never done anything like this in the lab before. It was a great learning experience. I learned that less is always more. It was very time consuming and I used a lot of growth media. I had to change the media every two days.
Experimental setup under sterile conditions
I count the cells using a Countess II Automated Cell Counter. It's no longer available for purchase through ThermoFisher Scientific. Only the fluorescent version and the new Countess 3 is available. We might be getting the new one. It's fluorescent as well. Yay!
Countess II Automated Cell Counter
Unfortunately my first attempt did not make any sense. The cells treated with 1.0 mg/mL had actually grown and the 50 ug/mL had the most dead cells. I know what you're thinking. I thought of it too. I wondered if I had gotten the antibiotic tubes backward. The curve was all over the place though and looked like a rollercoaster. I was also very meticulous about everything. This was important. I honestly don't think I made a mistake. But, then again, I've done some pretty dumb stuff in the lab!
So, I shall repeat the kill curve soon, with fewer antibiotic doses. I've been doing a lot of reading and looked at multiple protocols. It looks like I should be using a range from 100-800 ug/mL for this cell type. The range I used was based on this protocol. I started with 2.50 X 10^5 cells. I'm thinking I should start with twice as many cells. Or maybe I should start with less?
Science is unpredictable sometimes. Eureka!!
Sources:
My brain. I have studied this cell line intimately
A summary of HL-60 cells and how they are used for cell biology studies