Every small step...

The cells are okay! They’re not growing nearly as quickly as my guide’s. But it’ll do. Somehow, I already feel an attachment of sorts to them. That I’m responsible for them. Yes, I know it sounds lame, and very unprofessional. I guess I’m just completely caught up in the rush of it all. New lab. New equipment. The gloves and extra sterility. Plus, it’s been a full year since I worked with cells of any kind. And those were bacterial cells [S.aureus, to be precise. They’re the bacteria you find in pimples :D]

After checking them, we changed the medium in the plates. The cells should have fresh nutrition available now. Remember, TLC.

Oh, and in case you’re interested, here’s the protocol to dilute (or split) a culture of 293T. Note that I’m using 10ml of medium and 10cm dia. plates.

1) Aspirate (remove using a vacuum pump) the media using vacuum
2) Rinse with 5ml PBS (Phosphate Buffer Saline) gently
3)Aspirate PBS. Add 2ml trypsin. Leave for max. 2 mins. (This is to loosen the cells from the plate. Trypsin is an enzyme that digests protein)
4) Add 5ml of medium to stop the reaction.
5) Loosen the cells by tapping the side of the plate
6) Transfer 7ml of medium + trypsin to a 50 ml Falcon tube
7) Centrifuge for 10 mins at 1000 rpm, 25°C . Use balance.
8) Aspirate supernatant.
9) Add 1 ml medium to tube. Re-suspend cells.
10) Add 10ml of medium to fresh petri dishes. Add required dilution of re-suspended cells.
11) Move cells around in plate to spread evenly. Keep in incubator.

And that’s how it’s done :)