Klapperich Lab:Splitting IMR90 Cells
Replace this sentence with a brief description of the protocol and its goal.
- Put supplemented media, PBS and Trypsin/EDTA solution in the 37C water bath to heat up.
- Look at cells in the optical scope to make sure that they are 80% confluent before splitting.
- Aspirate old media
- Dispense 10 ml of PBS into the plate to wash the cells (it is important to remove the media, since the media inhibits trypsinization of the cells).
- Aspirate the PBS.
- Dispense 2 ml of Trypsin/EDTA solution into dish, make sure that the solution covers the bottom of the dish.
- Aspirate the Trypsin/EDTA solution after 15 seconds.
- Cover the plate with its lid.
- Place the plate in the incubator for 30 seconds – 1 minute.
- Remove plate back to biohood.
- Dispense 10 ml of media into the plate.
- Pipette vigorously up and down about 5 -10 times. (be careful not to get the media into the filter at the top of the pipet aid, also be careful about creating spray in the biohood)
- Look at the suspension in the optical scope to make sure that they are detached. They will look rounded and floating.
- Count the cells using the hemacytometer
- If you cannot count, assume that there are 250,000 cells/ml in your suspension.
- Take up the suspension into your pipette.
- Dispense 500,000 cells into each new plate you want to create (in the case when you cannot count, this is 2 ml of the suspension).
- Dispense 8 ml of fresh media into each new plate to bring the total media in each new plate to 10 ml.
- Gently tip the plates to distribute the cells evenly.
- Label the plates with the following:
- Cell type
- Population Doubling Number
- Replace the new plates in the incubator.
- Aspirate any leftover media from the old plates
- Bleach out the old plates and put them in the biowaste.
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
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Relevant papers and books
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4.
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56.
- Who has experience with this protocol?