Biomedical Engineering Reference
In-Depth Information
Method
1 Plate MEF cells on 0.1% gelatin-coated 24-well plates 48 h before starting to pick
colonies.
2Pre-warmES
+
G418 medium and 0.25% trypsin/EDTA at 37
◦
C. Thoroughly clean a
microscope, 200
μ
l pipette, pipette tip box and table with 70% ethanol.
r
3 Change the medium of a 24-well plate of MEF cells to ES
+
1
×
G418 medium.
4 Add 100
μ
l of trypsin/EDTA to each well in a few rows of a 96-well plate. This should
be repeated as necessary during picking.
5 Remove each plate of ES cell colonies from the incubator and circle all visible colonies
by holding the plate above your head and marking visible colonies on the
bottom.
s
,
t
6 Set a 20
μ
lpipetteto8
μ
l. Place a plate with circled colonies on the microscope stage.
To pick a colony, open the lid of the 10 cm plate with one hand and simultaneously
bring the pipette tip immediately adjacent to a colony. To pick the colony, gently
nudge the sides to loosen it from its MEF cell moorings and quickly aspirate, bringing
the colony with 8
μ
lofmedium.
7 Add the colony to a well of trypsin/EDTA in the 96-well plate and pipette up and down
a few times to help break up the colony. Start a countdown timer for 10min.
8 Repeat steps 6 and 7 as many times as possible inside the 10 min countdown.
u
9 After 10min, use a 200
μ
l pipette to vigorously pipette up and down each colony 10
times and then immediately transfer each trypsinized colony to a well of MEF cells in a
24-well plate (containing ES medium). Take care to also pipette up and down several
times at this stage to distribute the cells throughout the well.
v
10 After picking from a 10 cm plate is complete for the day, replace medium in the plate
with fresh ES medium
+
1
×
G418
+
1
×
FIAU.
11 48 h after picking, replace the medium in the 24-well plate with ES medium
+
1
×
G418 and then daily until harvesting.
w
Notes
r
If possible, it is preferable to use a microscope inside a culture hood since this will reduce the
possibility of culture contamination.
s
Different colored markers may be used for each day of picking, to avoid repeated picking of
the same colony. Picking will occur over four to five days.
t
Do not pick colonies that appear to be differentiating (poorly defined edges, very spread out,
less rounded cells).
u
Even if doing only a small number of colonies per picking cycle, a minimum of 3 minutes is
necessary to achieve good trypsinization.
v
Each individual well should be numbered in order to track the number of colonies picked.
w
Picked ES colonies will generally be ready for harvesting three to five days later, but please
note that exact harvesting time must be empirically determined by carefully monitoring cell
confluency in the 24 well plates on a daily basis.
