Klapperich Lab:Splitting IMR90 Cells

Overview

Replace this sentence with a brief description of the protocol and its goal.

Materials

• PBS
• Trypsin/EDTA

Procedure

1. Put supplemented media, PBS and Trypsin/EDTA solution in the 37C water bath to heat up.
2. Look at cells in the optical scope to make sure that they are 80% confluent before splitting.
3. Aspirate old media
4. 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).
5. Aspirate the PBS.
6. Dispense 2 ml of Trypsin/EDTA solution into dish, make sure that the solution covers the bottom of the dish.
7. Aspirate the Trypsin/EDTA solution after 15 seconds.
8. Cover the plate with its lid.
9. Place the plate in the incubator for 30 seconds – 1 minute.
10. Remove plate back to biohood.
11. Dispense 10 ml of media into the plate.
12. 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)
13. Look at the suspension in the optical scope to make sure that they are detached. They will look rounded and floating.
14. Count the cells using the hemacytometer
15. If you cannot count, assume that there are 250,000 cells/ml in your suspension.
16. Take up the suspension into your pipette.
17. 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).
18. Dispense 8 ml of fresh media into each new plate to bring the total media in each new plate to 10 ml.
19. Gently tip the plates to distribute the cells evenly.
20. Label the plates with the following:

• • Cell type
  • Population Doubling Number
  • Date
  • initials

1. Replace the new plates in the incubator.
2. Aspirate any leftover media from the old plates
3. Bleach out the old plates and put them in the biowaste.

Notes

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

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References

Relevant papers and books

1. 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. DOI:10.1073/pnas.78.11.6840 |
2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
3. ISBN:0879697164

Contact

• Who has experience with this protocol?