3-Splitting and passing HMT-3522 Cells

Feed cells with fresh media one day before splitting.

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 37^oC 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 mL of soybean trypsin inhibitor/25cm² and go to 8).

A) If cells do not detach, place flask back at 37^oC 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 ml and add 5 ml of tryoan blue, load 10 ml 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^oC over night.

B) Before plating cells in Vitrogen coated flask, aspirate Vitrogen mixture, wash once with DMEM/F12, then add warmed H14 medium.