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How to split cells

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Splitting Cell Cultures Grown in a T25 Flask.

PRIOR TO SPLITTING CELL CULTURE

Observe your culture with the inverted microscope and determine if the cells are either confluent or dense enough to split.

Remove tubes of DMEM media, Trypsin+EDTA, and PBS from the 4°C refrigerator and place them into a 37°C waterbath and allow to warm up.

Select new sterile T25 flasks and label them before placing them into the laminar flow hood.

Make sure you have enough 5 ml pipets and sterile aspiration pipets in the hood.

Spray the working surface in the hood with 70% ethanol and wipe it down.

Place an empty sterile 15 mL centrifuge tube in the hood for collecting cells into.

SPLITTING CELL CULTURE

Remove tubes of DMEM, Trypsin+EDTA, and PBS from the waterbath and dry them off. Spray them with 70% ethanol and place them into the hood.

Remove the cell cultures from the incubator and tighten the lids. Spray them with 70% ethanol and place them into the hood.

Remove the media from each cell culture flask by aspiration.

Wash cells with 5 mL of PBS, rock gently and then aspirate the PBS.

Add 0.6 mL of Trypsin+EDTA to each flask and rock gently to make sure that the bottom is completely covered. Tighten the lid. Incubate for 1.5 min and then bang the side of the flask sharply against the palm of your vertically held hand.

Add 4 mL of DMEM and pipet the media up and down, washing the interior of the flask so as to collect the maximum number of cells. Transfer to a sterile15 mL centrifuge tube.

Observe the flask that held the cell culture under the microscope and determine if the majority of the cells were released from the flask. If you have released the majority of the cells then discard this flask.

Centrifuge the 15 mL centrifige tube in the table top centrifuge for 5 min at setting 3.

Aspirate all the supernatant but avoid sucking up the cell pellet.

Resuspend the cells in 4-6 mL of DMEM by repeatedly drawing the media and cells up into the pipet and then expelling it until the cells are dispersed.

Add 1 mL of cell culture into new T25 flasks and add 4 mL of DMEM.

Place flasks back into the incubator (37°C and 5% CO2).

You should be able to observe that the cells have attached to the flask after a couple of hours.

AFTER THE SPLITTING IS COMPLETE

Return unused DMEM, Trypsin+EDTA, and PBS to the refrigerator. MAKE SURE THAT THE DOOR IS COMPLETELY SHUT!

Remove any other things that you have put into the hood.

Spray the working area in the hood with 70% ethanol and wipe it down.

Turn off the pipettor and aspirator pumps.

Close the hood sash and turn the light off. (Do not turn it to UV light!)

Put the cover back onto the 37°C waterbath.

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