11. On day 6 of culture, harvest the cells to evaluate the GFP
+
population using a flow cytometer (LSRII).
3.6 Clonal Isolation
by Limiting Dilution
1. Prepare a 96-well flat-bottom plate containing 100 μL of culture medium/well.
2. Transfer the GFP sorted cells to a 15-mL conical tube and
centrifuge at 500 Â g for 5 min at room temperature.
3. Discard the supernatant and resuspend the pellet with an
appropriate volume of culture medium to obtain the seeding
density of 5 cells/well.
4. Incubate the cells at 37
C, 5% CO 2 .
5. After 2 weeks, transfer the GFP
+ cells from the 96-well plate to
a larger well with the appropriate seeding density (Fig. 2b).
6. Evaluate the GFP
+ population using a flow cytometer (LSRII).
3.7 Immune Cell
Killing
To evaluate the cytotoxic activity of the NK-92 cell line (effector
cells), we co-culture the NK-92 cells with the GFP
+ K562 or Raji
cells at 5:1 (effector: target) ratio for 5 h. The cytotoxic activity can
be determined by tracking the GFP
+ /Zombie Aqua™
+ cells using
flow cytometer (Fig. 2c, d).
3.7.1 Co-culture
of the NK-92 Cells
with the GFP
+ K562 or
Raji Cells
1. Change the medium for the NK-92, GFP
+ K562, and Raji cells
24 h prior to co-culture.
2. The next day, perform cell counting using trypan blue exclusion assay to determine the cell number and viability.
3. Aliquot 3 Â 10
5 of the NK-92 cells and 6 Â 10
4 of the GFP
+
K562 or Raji cells into 15-mL tubes.
4. Centrifuge at 500 Â g for 5 min at room temperature.
5. Discard the supernatant and resuspend the pellet using 50 μL
of complete RPMI culture medium and transfer the cell suspension into 96-well plate.
6. Incubate the cells at 37
C, 5% CO 2 for 5 h.
7. Transfer the cells to a 5-mL round-bottom tube with a cap.
8. Centrifuge at 500 Â g for 5 min at room temperature.
9. Resuspend the pellet with 50 μL of FACS buffer.
10. Add 0.1 μL of Zombie Aqua™ Fixable Viability Kit, vortex
briefly, and incubate for 15 min in the dark.
11. Add 3 mL of FAC buffer, vortex briefly, and centrifuge at
500 Â g for 5 min at room temperature.
12. Discard the supernatant and resuspend the cells using 500 μL
of FAC buffer.
13. Immediately perform flow cytometry analysis.
226
Nontaphat Thongsin and Methichit Wattanapanitch
+
population using a flow cytometer (LSRII).
3.6 Clonal Isolation
by Limiting Dilution
1. Prepare a 96-well flat-bottom plate containing 100 μL of culture medium/well.
2. Transfer the GFP sorted cells to a 15-mL conical tube and
centrifuge at 500 Â g for 5 min at room temperature.
3. Discard the supernatant and resuspend the pellet with an
appropriate volume of culture medium to obtain the seeding
density of 5 cells/well.
4. Incubate the cells at 37
C, 5% CO 2 .
5. After 2 weeks, transfer the GFP
+ cells from the 96-well plate to
a larger well with the appropriate seeding density (Fig. 2b).
6. Evaluate the GFP
+ population using a flow cytometer (LSRII).
3.7 Immune Cell
Killing
To evaluate the cytotoxic activity of the NK-92 cell line (effector
cells), we co-culture the NK-92 cells with the GFP
+ K562 or Raji
cells at 5:1 (effector: target) ratio for 5 h. The cytotoxic activity can
be determined by tracking the GFP
+ /Zombie Aqua™
+ cells using
flow cytometer (Fig. 2c, d).
3.7.1 Co-culture
of the NK-92 Cells
with the GFP
+ K562 or
Raji Cells
1. Change the medium for the NK-92, GFP
+ K562, and Raji cells
24 h prior to co-culture.
2. The next day, perform cell counting using trypan blue exclusion assay to determine the cell number and viability.
3. Aliquot 3 Â 10
5 of the NK-92 cells and 6 Â 10
4 of the GFP
+
K562 or Raji cells into 15-mL tubes.
4. Centrifuge at 500 Â g for 5 min at room temperature.
5. Discard the supernatant and resuspend the pellet using 50 μL
of complete RPMI culture medium and transfer the cell suspension into 96-well plate.
6. Incubate the cells at 37
C, 5% CO 2 for 5 h.
7. Transfer the cells to a 5-mL round-bottom tube with a cap.
8. Centrifuge at 500 Â g for 5 min at room temperature.
9. Resuspend the pellet with 50 μL of FACS buffer.
10. Add 0.1 μL of Zombie Aqua™ Fixable Viability Kit, vortex
briefly, and incubate for 15 min in the dark.
11. Add 3 mL of FAC buffer, vortex briefly, and centrifuge at
500 Â g for 5 min at room temperature.
12. Discard the supernatant and resuspend the cells using 500 μL
of FAC buffer.
13. Immediately perform flow cytometry analysis.
226
Nontaphat Thongsin and Methichit Wattanapanitch