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.

Contact

 * Who has experience with this protocol?