This is not the greatest picture, but it was taken through an old light microscope with a cell phone camera for modern flair. These are HL-60 cells in RPMI media with 10% FBS and 1% Pen/Strep antibiotics. This is not the new high glucose media that I'm currently using. The new media is IMDM with 20% FBS and 1% antibiotics. I reduce the FBS to 10% two weeks after thawing. This picture shows cell growth at 90% confluency right before passaging to a new flask. I'm currently working on transfecting this cell line with a new method using Lipofectamine. I select for the transfected cells by adding Hygromycin B to the growth media. The plasmid includes a Hygromycin resistance gene. It seems to be working, but we shall see when I look on the fluorescent microscope tomorrow. I just finished mounting the cells on coverslips coated with Fibronectin.
It has been a wild week to say the least. We hadn't received blood samples since early December, due mostly to Covid and the Holidays. My boss was out sick this week as well, so all responsibility fell in my lap. We got 3 samples this week. Two sepsis control patients from the infectious disease clinic and one ICU sepsis patient. I received the sepsis sample yesterday. I was so tired yesterday afternoon that I screwed up on the PRR plate for the ICU patient, one of our most precious samples thus far. We just got approved for ICU patients last year. They were our first.
What is a PRR plate you ask? Well, I'll tell you... it's a pattern recognition receptor plate. We fluorescently label several common proteins on the surface of immune cells that recognize bacteria or fungus, as well as cell surface markers.
How did I mess up this said plate? Well, let me tell you, I have learned a very valuable lesson this week. As a matter of fact, I have learn a lot this week. In all aspects of my life. On blood days, I normally take care of isolating PBMCs (Peripheral Blood Mononuclear Cells) for protein assays. My boss isolates neutrophils and leukocytes. She does the PRR assay and the uptake assay (phagocytosis of Candia particles). I also do the ROS experiment and protein assay with neutrophils. There's more experiments but I don't have time to go into all that right now.
So, this PRR plate that I messed up, these plates are 96 wells with a notch in the bottom right. The plates that I use for PBMCs are different. They have a notch in the top left. So, I added fixative to the PRR plate after staining for the fluorescent antibodies. When the timer goes off, I remove the foil off the plate. I sit the plate down in the sterile hood. I look closely at the plate. This plate had been used 11 times before. I was using the last column for this precious sample. I had put the fix in the column on the other side. The side without cells!! I tried to salvage it by adding fixative and buffer. It was too late, the cells had dried up and were a blob. I threw the plate in biohazard waste. My tired brain was in autopilot and turned the plate so the notch was in the top left!
Lesson of the week: Look closely and attentively when you are doing something important.
I emailed our clinical coordinator this morning and they are going to have another vial pulled with the morning labs for the ICU sepsis patient. This does not normally happen with sepsis patients. They usually get discharged to go home after treatment. This patient is extremely sick so they will be in the hospital for a while. I only need 1mL of blood to redo the assay.
Until next time, fellow Hive peeps! Stay cool 馃槑
