I had to redo the transfection protocol in mid-April with the K-562 cells as none of the cells transfected in February and March grew. This time, I decreased the lipofectamine reagent by half. I had read that some cell types may be sensitive to the lipoprotein reagent during transfection. I thought it would be wise to try less. Less reagent proved to be successful because the newly transfected cells recently started to increase!
As always, the cells were resuspended in fresh IMDM media containing 200 ug/mL of Hygromycin B, 48 hrs after transfection. Hygromycin B is an antibiotic that inhibits grow of mammalian cells. Both the positive (+ protein) and negative (- protein) controls are transfected with a plasmid containing a Hygromycin resistance gene for selection along with a red fluorescent protein tag (RFP) for visualization. Only the cells that are successfully transfected should grow in media containing the antibiotic.
Around the end of April, I noticed significant cell death when I counted the cells. I salvaged the remaining cells and resuspended them in 1 mL of fresh IMDM media containing antibiotic. There were less than 1 X 10^6 cells left. I changed the media every few days and on May 10, I noticed the media changed to orange, indicating cell growth. Fresh media is a deep shade of reddish pink. Cellular growth reduces the pH and the color of the media changes. Yellow indicates high cell death or contamination. I counted them and added another milliliter of fresh media so they could continue growing in a 12-well plate.
By May 12, I had to transfer them to T25 flasks because they were rapidly increasing. On Monday, May 15, I had over 5 million cells for both the positive and negative control. I decided to split the cells and transferred some to T75 flasks so I could ramp up the cellular proliferation. The T25 flasks contained about 2 X 10^6 total cells. All flasks are growing well. My goal is the freeze back aliquots of each transfected cell culture. I also need to perform experiments with these cells to compare them to the sepsis patients and healthy donors in our clinical study.
I counted the cells growing in T25 flasks with our new COUNTESS 3 FL cell counter. The counter will detect the RFP tag on both the positive and negative control. Only the positive cells contain the genetic code for our protein of interest that will hopefully be overexpressed on the surface of the cells. We will detect this protein by Flow Cytometry. We discovered in our research that sepsis patients have an increased expression of this protein on their neutrophils when compared to healthy donors as well as infectious disease patients. We are trying to better understand this overexpression of protein as seen in septic shock.
As shown in the last two pictures, there were 1.18 X 10^6 total positive cells and 64% of them were expressing RFP. For the negative controls, there was a total of 2.51 X 10^6 cells and 45% of them were positive for RFP. I will count all of the flasks on Friday, May 19.
Thanks for reading my post. Enjoy the pics!
All photos were taken with my cell phone camera.
Passaged cells in T25 and T75 flasks
Preparing the cells for counting
Close-up of the counting slide
Positive transfected cells
Negative transfected control cells
