RRC-TPS: Preparing ES Cells for Microinjection: Protocol
Your clones will be injected over a 2- to 3-day period (T, W, Th). The number of embryos injected on a given day will vary, depending on the embryo yield. Plan to give us one or two clones per day.
Following thawing ES clones should be passaged (split) one or two times before injection day. If colonies of some clones are few or not growing well you should still trypsinize the cells and then plate them onto a fresh dish/well. Confluent or fast-growing cells may need to be split the day after initial plating. The final passage should be onto wells of a 12-well plate (see note). In our experience clones that are split the day before injection yield the most/best chimeras.
Have a choice of several wells available on each injection day, i.e., it is a good idea to plate different dilutions (densities) of each clone when passaging onto the 12-well plate (see note).
On the day of injection (plan to get your cells to us at 12:30):
- Choose subconfluent (80-85%, colonies are nearly touching each other) wells for injection.
- Rinse with PBS. Add 300µl trypsin, place in incubator for 5-10 min.
- Add one-to-two volumes of medium; pipet up-and-down or gently rock plate to mix.
- Using a yellow tip attached to a 1-ml serological pipet, gently pipet up-and-down several times to generate a single-cell suspension. Monitor your progress under microscope (see note).
- Place cells in 15-ml conical tube; rinse the well with additional 1ml medium, add to cells in tube.
- Spin; aspirate, leaving a small volume of medium (~1-2X vol. of the packed pellet).
- Flick the tube to resuspend the pellet in the remaining medium.
- Add 2ml ES medium (no selective drugs, no LIF) supplemented with 20mM HEPES (see note). Place tube on ice.
- You may use larger wells or dishes if you prefer, but a single well of a 12-well plate yields plenty of cells for us to work with.
- It is important that the ES colonies are dispersed into single cells (no clumps) following trypsinization.
- If your collected ES cells are not in medium supplemented with 20mM HEPES, please let us know – we will add HEPES as soon as the cells arrive.