Purpose
To describe the maintenance of cells in culture.
Safety
Equipment
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Laminar flow hood
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CO2 Incubator
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Mechanical Pipettor
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Inverted microscope
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Vacuum pump and flask
Materials
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Complete cell culture medium, appropriate for the cell line
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Tissue culture flasks of appropriate sizes
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Tissue culture plates - 96 well or 24 well.
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Sterile pipets, assorted sizes
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Multichannel pipet and sterile tips.
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Pasteur pipets, 9¡±, sterile, unplugged.
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70% alcohol
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Sterile Petri dishes.
Procedure
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General considerations
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Turn on the hood and allow to run for at least 10 minutes before starting.
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Pre-warm all media in 37o C water bath.
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Wipe all surfaces with 70% alcohol before starting.
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For cells in flasks.
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Cells are split or refed every 3-4 days.
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Remove the flasks from the incubator. Examine flasks with inverted microscope.
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Place flasks and medium under hood.
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Remove an aliquot of the cell suspension for counting with a hemocytometer (SP 05-009) and viability determination (SP 09-005).
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Label the appropriate number of new flasks with the cell line name, the passage, the slit ratio or seeding density and the date.
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Aseptically transfer the required number of cells to the new flask. Add fresh medium to the flask.
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For T-25 flask - maximum of 10 ml of medium.
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For T-75 flask - maximum of 50 ml of medium.
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For T-150 flask - maximum of 100 ml of medium.
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For T-225 flask - maximum of 200 ml of medium.
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Recap the flask(s), gently shake to evenly disperse the cells.
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Return the flasks to the incubator. Loosen the caps 1/2 turn, if necessary.
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For 96 well plates.
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Plates are re-fed every 3-4 days.
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Remove the plates from the incubator, and examine microscopically or with the mirror. Mark wells that are positive for cell growth.
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Place plates and medium under the hood.
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If supes are being collected for an assay, pre-label the plates for the supernatants.
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Transfer 150 ml of supernatant to the clean, labeled plate.
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Store all supernatant plates at 2-80 C until ready to assay.
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Remove spent medium with a sterile Pasteur pipet attached to a vacuum. Hold the pipet at a 45 degree angle about 1/2 way down the well.
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Start at the top left corner of the plate and work back and forth across the wells to the lower right corner.
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Repeat with all plates.
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Aseptically transfer medium into sterile Petri dish. Add 150-175 ml of fresh medium into each well using a multichannel pipettor. Work from the end of the plate furthest from your hand, to minimize the number of times your hand passes over the plate.
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Repeat with all plates. Add additional medium to a new, clean Petri dish if more is needed. Change pipet tips for each plate.
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Return the plates to the incubator.
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For 24 well plates
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Remove plates from the incubator and examine microscopically.
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Place plates and medium under the hood.
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If supes are being collected for an assay, pre-labels the plates for the supernatants.
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Transfer 1-2 ml of supernatant to the clean, labeled plate.
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Store all supernatant plates at 2-8o C until ready to assay.
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Remove spent medium with a sterile Pasteur pipet attached to a vacuum. Hold the pipet at a 45 degree angle about 1/2 way down the well.
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Start at the top left corner of the plate and work back and forth across the wells to the lower right corner.
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Repeat with all plates.
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If cells need to be split, indicated by orange colored medium and the cells covering more than half the surface of the well, add 2 ml of fresh medium to the well.
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Add 2 ml of fresh medium to each well.
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Return the plates to the incubator.
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Transferring cells from a 96 to 24 well plate.
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Identify the clones to be expanded.
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Using a sterile pipet tip and pipettor, resuspend the cells by pipetting.
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Transfer 150 ml of the cell suspension one well of a pre-labeled 24 well plate.
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NOTE: Use only the top row of each 24 well plate for new clones to allow room to expand the cells down the plate as they grow.
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Add 2 ml of fresh medium to each well of the 24 well plate. Add 150 ml of fresh medium to each 96 well.
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