3.8 Embryo
Dissociation
It is important that once the embryos have been dissected cells are
dissociated, sorted and frozen on dry ice in less than 1 h.
1. Defrost all solutions (FACsMax solution, FBS, trypsin, etc.) at
4
C overnight and keep on ice when starting the experiment.
Pre-cool a table top centrifuge to 4
C.
2. Take the photoconverted embryos out of the methyl cellulose
by delicately flowing E3 with a plastic pipette over the
embryos. Transfer the embryos into fresh E3 + tricaine
medium.
3. Using n3 tweezers, manually dissect the head and yolk from the
tail (to be dissociated). Using one pair of forceps, pinch right
under the yolk at the beginning of the yolk stalk, and with the
second forceps cleave the tail away.
4. Immediately transfer the tail to a cooled 1.5 mL tube on ice
using a long glass Pasteur pipette carrying over as little E3 as
possible. Aim to a minimum of 10–20 embryos per tube.
5. Spin down the embryos using the pre-cooled 4
C centrifuge at
400 Â g for 1 min.
6. Remove the supernatant under a dissecting microscope, be
careful not to disturb the pellet.
7. Add 200 μL of trypsin-EDTA. Wash a P200 tip in sterile PBST,
by suctioning and discarding the solution. Proceed to dissociate the pellet by vigorously pipetting up and down for 10 min
or longer until no clumps of tissue remain.
8. Add 5% of HS-FBS to stop the reaction.
9. Spin down in pre-cooled 4
C centrifuge at 400 Â g for 5 min.
10. Remove supernatant under dissection microscope, be careful
not to disturb the pellet.
11. Slowly add 200 μL of FACsMax solution without disturbing
the pellet. Spin down in pre-cooled 4
C centrifuge at 400 Â g
for 5 min.
12. Remove supernatant under dissection scope, be very careful
not to disturb the pellet and repeat steps 10–12.
13. Resuspend the dissociated cell pellet in 300 μL of FACsMax
solution using PBST washed tips. Transfer into 35 μm filtered
FACS collection tube and keep on ice.
14. Proceed immediately to the FACS machine. Gating conditions
depend on the sorter used and should be experimentally determined. At the end of sorting, samples can be immediately
processed or frozen at À80
C.
102
Zain Alhashem et al.
Dissociation
It is important that once the embryos have been dissected cells are
dissociated, sorted and frozen on dry ice in less than 1 h.
1. Defrost all solutions (FACsMax solution, FBS, trypsin, etc.) at
4
C overnight and keep on ice when starting the experiment.
Pre-cool a table top centrifuge to 4
C.
2. Take the photoconverted embryos out of the methyl cellulose
by delicately flowing E3 with a plastic pipette over the
embryos. Transfer the embryos into fresh E3 + tricaine
medium.
3. Using n3 tweezers, manually dissect the head and yolk from the
tail (to be dissociated). Using one pair of forceps, pinch right
under the yolk at the beginning of the yolk stalk, and with the
second forceps cleave the tail away.
4. Immediately transfer the tail to a cooled 1.5 mL tube on ice
using a long glass Pasteur pipette carrying over as little E3 as
possible. Aim to a minimum of 10–20 embryos per tube.
5. Spin down the embryos using the pre-cooled 4
C centrifuge at
400 Â g for 1 min.
6. Remove the supernatant under a dissecting microscope, be
careful not to disturb the pellet.
7. Add 200 μL of trypsin-EDTA. Wash a P200 tip in sterile PBST,
by suctioning and discarding the solution. Proceed to dissociate the pellet by vigorously pipetting up and down for 10 min
or longer until no clumps of tissue remain.
8. Add 5% of HS-FBS to stop the reaction.
9. Spin down in pre-cooled 4
C centrifuge at 400 Â g for 5 min.
10. Remove supernatant under dissection microscope, be careful
not to disturb the pellet.
11. Slowly add 200 μL of FACsMax solution without disturbing
the pellet. Spin down in pre-cooled 4
C centrifuge at 400 Â g
for 5 min.
12. Remove supernatant under dissection scope, be very careful
not to disturb the pellet and repeat steps 10–12.
13. Resuspend the dissociated cell pellet in 300 μL of FACsMax
solution using PBST washed tips. Transfer into 35 μm filtered
FACS collection tube and keep on ice.
14. Proceed immediately to the FACS machine. Gating conditions
depend on the sorter used and should be experimentally determined. At the end of sorting, samples can be immediately
processed or frozen at À80
C.
102
Zain Alhashem et al.