Biomedical Engineering Reference
In-Depth Information
PROTOCOL 10.16
Harvesting ES Cell Colonies
Equipment and reagents
•
Incubator at 37
◦
Cwith5%CO
2
•
Water bath at 37
◦
C
•
FBS (heat inactivated)
•
0.25% trypsin with EDTA
•
1
×
Dulbecco's PBS
•
DMSO
•
Cryotubes.
Method
1 Thaw heat-inactivated FBS and pre-warm 0.25% trypsin/EDTA to 37
◦
C.
2 Select which wells of the 24-well plates containing ES cells that are ready for
harvesting.
x
3 Fully aspirate the medium from 15 wells that are to be harvested. Add 350
μ
l of 0.25%
trypsin/EDTA to each well and put the plate in the 37
◦
C incubator for 5 min.
4 During the 5 min incubation, label two sets of 15 tubes, both from 1 to 15 (16 to 30 in
second round, etc.).
y
5 After the 5 minute incubation, add 400
μ
l of FBS to each trypsinized well. Pipette up
and down 15 times to dissociate cells.
6 Put 400
μ
l of cells into a tube for freezing at
−
80
◦
C and the remaining 350
μ
l into the
other tube for PCR screening.
7 To each of the tubes destined for
−
80
◦
C freezing, add 44
μ
lofDMSOandmix
immediately and thoroughly.
z
,
aa
8 Spin the tubes destined for PCR screening at maximum speed for 2 min in a bench-top
centrifuge and aspirate the medium. The pellets should then be stored at
−
80
◦
Cin
boxes separate from those containing cells frozen in FBS/DMSO.
9 Repeat steps 3-8 until all colonies are ready to be harvested that day have been
frozen.
Notes
x
A well that is ready will generally have 20-100 medium to large sized colonies per well.
y
This will allow the preservation of half the cells (first set) for injection and the digestion of
the other half (second set) for identification of positive clones by PCR.
z
These must be stored at
−
80
◦
C immediately. The tubes for PCR can be temporarily stored at
4
◦
C for a few rounds of harvesting.
aa
It is helpful to write down the last number harvested before beginning the next round so as
to correctly number the tubes in the proper, sequential manner.
