TissueCulture:splitting cells: Difference between revisions
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==Procedure== | ==Procedure== | ||
As per t175 dish | |||
#Take the stock plate from the incubator | #Take the stock plate from the incubator | ||
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#Add 10 ml of DMEM+FBS+glutamine+antibiotics to a new, labelled petri dish | #Add 10 ml of DMEM+FBS+glutamine+antibiotics to a new, labelled petri dish | ||
#Add 1 ml of the trypsinized cells to the new dish, discard the rest (aspirate off then throw away dish) | #Add 1 ml of the trypsinized cells to the new dish, discard the rest (aspirate off then throw away dish) | ||
# | #Incubate at 37°C until desired confluency | ||
==To add== | |||
DMEM needs 50 ml FBS, 5 ml glutamine, 5 ml antibiotics | |||
The final concentration of FBS should be 10% v/v | |||
The final concentration of antibiotics should be 1% v/v | |||
Use this media for everything except lipofectamine transfection | Use this media for everything except lipofectamine transfection | ||
Low serum media w/o antibiotics, such as Opti-MEM, should be used during transfection, and transduction. | |||
You can use Hela most of the time, but Heck is also good? | You can use Hela most of the time, but Heck is also good? | ||
Latest revision as of 14:05, 20 January 2014
Concept
Tissue culture cells need to be "split" to keep them alive. The process involves taking a portion of the cells and resuspending them in fresh culture media so they can grow. If they're left too long in the culture media, they will die, so this process needs to be repeated to keep them alive. They're kept in DMEM, which is red in color. Once it turns orange, the cells are ready to be split again. The DMEM must be used at room temperature or, optimally, at 37°C
Procedure
As per t175 dish
- Take the stock plate from the incubator
- In the tissue culture hood and being careful not to touch the cells (on the bottom), aspirate off all the media (use a clean tip!)
- Add 10 ml of PBS carefully (so as not to kill the cells, use the "gravity" setting on the pipettor)
- Add 500 ul of trypsin-EDTA
- Swirl around
- Put at 37°C for 5 minutes to let the trypsin work
- Add 10 ml of DMEM+FBS+glutamine+antibiotics to the trypsinized cells
- Add 10 ml of DMEM+FBS+glutamine+antibiotics to a new, labelled petri dish
- Add 1 ml of the trypsinized cells to the new dish, discard the rest (aspirate off then throw away dish)
- Incubate at 37°C until desired confluency
To add
DMEM needs 50 ml FBS, 5 ml glutamine, 5 ml antibiotics
The final concentration of FBS should be 10% v/v The final concentration of antibiotics should be 1% v/v
Use this media for everything except lipofectamine transfection Low serum media w/o antibiotics, such as Opti-MEM, should be used during transfection, and transduction.
You can use Hela most of the time, but Heck is also good?
