11. After completion, carefully remove the Nucleocuvette™ vessel
from the retainer.
12. Resuspend the cells with 400 μL of culture medium and mix
the cells by gently pipetting up and down for two to three times
using the supplied pipette.
13. Carefully transfer 500 μL of the cell suspension into the 6-well
plate containing pre-incubated culture medium (step 2).
14. Incubate the cells at 37
C, 5% CO 2 .
3.4.2 Evaluation of GFP
Knock-in Cells
The following day, the expression of GFP protein can be observed
under a fluorescence microscope or determined using a flow cytometer. Transient GFP protein expression should be monitored
due to the presence of non-integrated GFP donor plasmid in the
transfected cells without the HDR (see Note 11).
1. Harvest 1 Â 10
5 transfected K562 or Raji cells to a 5-mL
round-bottom tube with a cap.
2. Centrifuge at 500 Â g for 5 min at room temperature.
3. Discard the supernatant and resuspend the pellet with 50 μl of
FAC buffer.
4. Add 0.1 μL of Zombie Aqua™ Fixable Viability Kit, vortex
briefly, and incubate for 15 min in the dark.
5. Add 3 mL of FAC buffer, vortex briefly, and centrifuge at
500 Â g for 5 min at room temperature.
6. Discard the supernatant and fix the cells using 500 μL of 1%
paraformaldehyde, the stained cells can be stored at 4
C for up
to 2 weeks.
7. Determine the GFP-positive population using a flow cytometer
(LSRII) and analyze using the FlowJo software. Exclude the
dead cells, which stained positive for Zombie Aqua™.
3.5 Enrichment
of the GFP
+ K562
and Raji Cells Using
FACS
To enrich the GFP
+ cell population of the transfected K562 and
Raji cells, we use FACS to sort the GFP
+ cells. In some occasions,
the GFP
À cells potentially contaminate the GFP
+ cells after
FAC sorting. Further enrich the sorted cells using FACS or limiting
dilution (Subheading 3.6) to obtain high purity of GFP
+ cells
(Fig. 2a).
1. Harvest 4 Â 10
6 transfected K562 or Raji cells to a 15-mL
conical tube and centrifuge at 500 Â g for 5 min at room
temperature.
2. Discard the supernatant and resuspend the cell pellet with 1 mL
of FACS buffer.
3. Filter the cell suspension using a 5-mL round-bottom tube
with a strainer cap.
224
Nontaphat Thongsin and Methichit Wattanapanitch
from the retainer.
12. Resuspend the cells with 400 μL of culture medium and mix
the cells by gently pipetting up and down for two to three times
using the supplied pipette.
13. Carefully transfer 500 μL of the cell suspension into the 6-well
plate containing pre-incubated culture medium (step 2).
14. Incubate the cells at 37
C, 5% CO 2 .
3.4.2 Evaluation of GFP
Knock-in Cells
The following day, the expression of GFP protein can be observed
under a fluorescence microscope or determined using a flow cytometer. Transient GFP protein expression should be monitored
due to the presence of non-integrated GFP donor plasmid in the
transfected cells without the HDR (see Note 11).
1. Harvest 1 Â 10
5 transfected K562 or Raji cells to a 5-mL
round-bottom tube with a cap.
2. Centrifuge at 500 Â g for 5 min at room temperature.
3. Discard the supernatant and resuspend the pellet with 50 μl of
FAC buffer.
4. Add 0.1 μL of Zombie Aqua™ Fixable Viability Kit, vortex
briefly, and incubate for 15 min in the dark.
5. Add 3 mL of FAC buffer, vortex briefly, and centrifuge at
500 Â g for 5 min at room temperature.
6. Discard the supernatant and fix the cells using 500 μL of 1%
paraformaldehyde, the stained cells can be stored at 4
C for up
to 2 weeks.
7. Determine the GFP-positive population using a flow cytometer
(LSRII) and analyze using the FlowJo software. Exclude the
dead cells, which stained positive for Zombie Aqua™.
3.5 Enrichment
of the GFP
+ K562
and Raji Cells Using
FACS
To enrich the GFP
+ cell population of the transfected K562 and
Raji cells, we use FACS to sort the GFP
+ cells. In some occasions,
the GFP
À cells potentially contaminate the GFP
+ cells after
FAC sorting. Further enrich the sorted cells using FACS or limiting
dilution (Subheading 3.6) to obtain high purity of GFP
+ cells
(Fig. 2a).
1. Harvest 4 Â 10
6 transfected K562 or Raji cells to a 15-mL
conical tube and centrifuge at 500 Â g for 5 min at room
temperature.
2. Discard the supernatant and resuspend the cell pellet with 1 mL
of FACS buffer.
3. Filter the cell suspension using a 5-mL round-bottom tube
with a strainer cap.
224
Nontaphat Thongsin and Methichit Wattanapanitch