HL-60 cells are grown in suspension and therefore must be treated differently to (the more common) adherent cultures. In order to keep the cells alive and growing well, the culture medium must be changed regularly. This allows nutrients and growth factors to be replenished and the cells to be re-seeded to a concentration which minimises overcrowding and cell death. From previous studies we found that the HL-60 cell line grows favourably at around 1×105 cells/ml. Therefore, we typically re-seeded the cells at this density every 2 – 3 days.
Corinne, Luke and Lee-Anne observing the HL-60 cells prior to seeding
The HL60 cells are kept in an incubator at 37°C and 5% CO2. Before seeding, the cells must be checked under a light microscope for morphological changes which would suggest appropriate growth and concentration of cells.
HL60 cells under a light microscope, prior to reseeding.
To avoid any risk of infection (bacteria, mycoplasma or fungi) to the cells, all cell culture work must be carried out under sterile conditions in a cell culture hood, ensuring thorough that aseptic techniques are used on every occasion the cells are handled.
To re-seed the cells, firstly the culture must be centrifuged to separate the medium and cells, as the cells will form a pellet in the bottom of the tube.
The cell culture underwent centrifugation for 10 minutes at 1200 rpm
Following centrifugation, the supernatant is poured off and the cells re-suspended in fresh, warmed RPMI medium. A cell count is then performed using a TC20 automated cell counter. The cells are then diluted to the concentration wanted for growing them (1×105 per ml). 50μl of cells and 50μl of Trypan Blue stain were mixed to be pipetted into a cell counter slide and used in the cell counter in order to both count the cells and to ensure that the cells were at least 95%, as only dead cells allow the Trypan Blue to enter them, and this effect is used by the TC20 cell counter to calculate viability.
BioRad TC20 Automated Cell Counter counting the number of live HL-60 cells in our culture
The T75 (75 cm2) cell culture flask with the new medium and correct amount of cells is then placed into the incubator.