Splitting and Passing HMT-3522 Cells
The number of cells is very important when considering whether to plate them or wait until a certain day to use them. center your cell culture work around how you expect your cells to grow.
Feed cells with fresh media one day before splitting.
1. Aspirate media
2. Wash 1 1/2 ml of fresh 0.25% trypsin, then add 1 ml trypsin and stick in incubator for 3 min.
incubate time = 3 min for T4, 5 min for S1, 4 min for S2 cells. T4 cells- timing is very important. put timer on! trypsinizing the cells for too long can lead to cells changing morphology.
3. Add 95 microliters of soybean trypsin inhibitor. slam these cells very hard- they cannot be in trypsin for too long!
4. Immediately add 10 ml media to stop and dilute reaction
5. Pipette up and down to mix the cells
HMT3522 S1 cells are ready to split once 2D colonies look like rounded islands (that is, the edges of the colony become smooth).
This is usually when the plate is approximately 60% confluent and can be anywhere from day 6 to 10 after plating.
If they are higher than passage 34, S1’s are plated directly onto plastic.
If lower than passage 34, S1’s are plated on Vitrogen (collagen 1) coated plates.
HMT3522 T4-2 cells are split when they reach 80% confluency and are plated on Vitrogen (collagen 1) coated plastic (SEE VITROGEN COATING FLASK PROCEDURE)
1) Aspirate H14 medium
2) For every 25 cm2 surface area of plate, add 0.5 to 1 ml of warmed 0.25% trypsin-EDTA (room temp, ask Eva Lee or Hong for more details)
3) Aspirate trypsin
4) Add back 1/3 amount of 0.25% trypsin as was added in step 2 (commonly 1ml for T75 flask)
5) Place flask in 37oC incubator for 3 to 6 minutes
6) Briefly remove flask from incubator and gently knocks the cells to help them to get off as soon as possible
7) If cells detach, immediately add warmed H14 media and 60 μL of soybean trypsin inhibitor/25cm2 and go to 8)
A ) If cells do not detach, place flask back at 37oC for one minute. (S1 cells are not usually detached yet)
B ) Again remove from incubator and tap flask (to detach cells) and redistribute trypsin (Most cells detach by this time, although sometimes S1 cells need more incubation time)
C) If more time is needed, repeat steps (7A) and (7B)
8) Wash cells down into the medium
9) Pipette mixture up and down three to five times to dissociate cell aggregates
10) Transfer mix to a 15 ml Falcon
11) Spin down
12) Resuspend cells in fresh complete media (H14)
11) Count cells
- take an aliquot of 95 μl and add 5 μl of tryoan blue, load 10 μl on an hemacytometer. Count the cells and multiply by 10 000 to have the number of cells per ml
12) Plate HMT 3522 S1 cells at a density of 2x104 cells/cm2 on plastic and HMT 3522 T4-2 cells at a density of 1X104 cells/cm2 onto vitrogen coated plates- For 3d or OT culture, see the protocols
A ) To vitrogen coat, add 1 ml Vitrogen to 44 ml of cold 1X PBS and store at 4 C over night
B ) Before plating cells in Vitrogen coated flask, aspirate Vitrogen mixture, wash once with DMEM/F12, then add warmed H14 medium