How to count and how to pellet COS cells?

Equipment

1 x 5ml aliquot of Trypsine Versine (TV)

1 x 800ul PBSa

1 x glass beaker (250ml) for waste

2x Universals (one to use as balance)

Tissue culture hood

Foetal Calf Serum (FCS)

ICE- to put the cells on if you need to leave briefly

Starting materials

1 flask of COS cells seeded 72 hours ago in a T75 (75cm²). Seeded with 10⁶ COS cells. Grown at 37 �C 5% CO₂, 100% humidity. Grown in DMEM and Glutamax and 10% Feotal Calf Serum (FCS).

To begin, open bottle lids and have them ready e.g. open PBSa lid.

1. Unscrew cap on culture flask. Pour off medium that the cells have been growing in. Pour into glass beaker.
2. Squirt 15mls of PBSa into culture flask. Do not squirt it directly at the cells. (NOTE: only touch the top of the glass pipettes, avoid touching the tip or lower regions onto anything unnecessary or non-sterile.)
3. Screw cap back onto culture flask. Lay the culture flask flat, and swish the liquid around to wash cells. Do not let liquid go into the cap area or make it wet.
4. Repeat steps 1 to 3 so that the cells are washed in PBSa for a total of 3 times.
5. Pour off the PBSa after the 3^rd wash.
6. Pour in the 5ml aliquot of TV without touching the aliquot tube against the culture flask.
7. Put the cap back onto the culture flask. Distribute the TV evenly over the cells by swishing the liquid around. Do not let liquid get into the cap.
8. Pour off most of the TV so that 1-2ml is left. This should be enough to lightly cover the whole of the layer of cells on the bottom of the flask.
9. Incubate the culture flask in the incubator for a few minutes. The raised temperature will speed up the action of the trypsin.
10. Put the culture flask back into the hood. Holding it flat on the bottom of the hood, hit it several times against the air flow grille at the front of the hood. This should dislodge the cells. The cells should start to form into balls. You can now look at it under a microscope to confirm that the cells are lifting off the bottom of the tissue culture dish.
11. Add 10ml of DMEM with 10% FCS to the culture flask to neutralize the trypsin. (NOTE: 10ml of medium should be adequate to neutralize 1-2ml of trypsin.)
12. Pipette the mix up and down to break up any clumps of cells. Suck up most of the mix into the pipette, place the tip in the corner, then expel, so that the cells are under pressure as they are ejected.
13. Pipette the cells into one universal, and fill the other with an equal amount of water (to act as a balance).
14. Take 20ul out of the mix to count whilst doing the centrifuging.
15. Spin the universals at 1000rpm for 5minutes at 4 �C.
16. Whilst the cells are spinning, put 16ul of the sample into the haemocytometer (it will fill both sides), and count the cells. Calculate the concentration of the cells. (We had 8.7x10⁶ cells which will be resuspended in 5ml to give 5x 1ml of 1.7x10⁶ cells.)