HAP1 cells are cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with 10% FBS and 1% Pen/Strep. Always be gentle when resuspending cells to avoid any mechanical stress. Each knockout cell line may grow at different doubling times and cells may have a slightly different morphology.
- Iscove’s Modified Dulbecco’s Medium (IMDM):
- GIBCO, Cat.No. 12440-053 (500ml)
- Fetal Bovine Serum: Sigma Aldrich, Cat.No. F2442-500ML
- Trypsin-EDTA Solution (1X): GIBCO, Cat.No. 25300096 (100 ml)
- Penicillin / Streptomycin: GIBCO, Cat.No. 15140-122 (100 ml)
Media for Freezing
Medium A: IMDM + 20 % FBS
Medium B: IMDM + 20 % FBS + 20 % DMSO
Thawing HAP1 Cells
- Thaw the vial with frozen cells quickly. You may do this by placing the vial in the 37 °C waterbath.
- Dilute cells in 10 mL of pre-warmed culture medium.
- Optional: Spin down cells for 5 minutes at 1200rpm/300 x g. Aspirate medium without disturbing the pellet and add 10 mL fresh medium.
- Transfer cells to a 10 cm dish.
- Monitor cells closely for the next 2 days.
- Change medium after 24 hours.
- Please see the pictures below for examples of how cells will look 48 hours after thawing.
Figure 1. HAP1 clones 48 hours post-thawing
Passage / Culture of HAP1 Cells
- Aspirate medium from cells.
- Wash with PBS to remove all traces of medium and FBS.
- Add Trypsin (0.05 %) and incubate at 37 °C until cells begin to detach (usually 3 to 5 minutes).
- Add medium to stop trypsinization. Resuspend cells and transfer desired amount of cells to a new dish.
Note: HAP1 cells should be split 1:10 to 1:15 every 2 to 3 days. Keep in mind that growth rates may vary between different clonal HAP1 cell lines. HAP1 cells should never be kept at a high density (maximum density: 75 % confluency). See pictures below for examples of low density and high density HAP1.
Freezing HAP1 Cells
- Trypsinize and spin down cells for 5 minutes at 1200rpm/300 x g.
- For freezing cells, use a 1:1 mixture of Medium A: Medium B. First, resuspend cell pellet in Medium A.
- Slowly add Medium B.
- Transfer cell suspension to suitable cryovials.
- Be sure to transfer the cryovials to the freezer within five minutes after addition of DMSO-containing medium.Place tubes in a suitable freezing container in a – 80 °C freezer in order to allow slow cooling.
- The following day, transfer cells to liquid nitrogen.
Note: Using Medium A and B allows more flexibility when preparing many samples for freezing. If few samples are being prepared, use a complete freezing medium consisting of IMDM + 20 % FBS + 10 % DMSO. After step 1, resuspend cells 1 mL of complete freezing medium and continue to step 4.
Figure 2. HAP1 cells at low density (left panel) and high density (right panel)