3 Methods
The following section describes a general protocol that we use for
site-specific integration of the GFP gene in cell lines for tracking
functional immune cell killing (Fig. 1). The protocol includes
culture of the K562, Raji, and NK-92 cell lines, preparation of
individual plasmids containing sgRNA, Cas9 and GFP genes, transfection of the plasmids into the cell lines, enrichment of the GFP
+
cells by FACS and limiting dilution. After clonal expansion, the
engineered GFP
+ K562 or Raji cells can be used as target or
non-target cells for the NK-92 cell killing, respectively. This
approach facilitates immune cell killing assay since we can easily
track and directly evaluate the efficiency of immune cell cytotoxic
function.
3.1 Culture of K562
and Raji Cell Lines
3.1.1 Thawing
1. Warm K562 and Raji cell culture medium to room
temperature.
2. Quickly thaw the cryopreserved K562 and Raji cells in a 37
C
water bath until only a small ice crystal remains.
3. Wipe the outside of the cryovial with 70% ethanol.
4. Gently pipette the cell suspension and transfer to a 15-mL
conical tube containing 9 mL of RPMI-1640 basal medium.
5. Centrifuge the cells at 500 Â g for 5 min at room temperature.
6. Discard the supernatant and resuspend the pellet in 5 mL of
culture medium.
7. Transfer the cell suspension into a 25-cm
2 cell culture flask and
incubate at 37
C, 5% CO 2 (see Note 1).
3.1.2 Passaging
Passaging is performed when the cells reach a maximum density:
K562 cell line, 1 Â 10
6 viable cells/mL, and Raji cell line, 3 Â 10
6
viable cells/mL.
1. Gently resuspend the cells using a serological pipette and transfer the cell suspension to a 15-mL conical tube.
2. Aliquot a small amount of the cell suspension into a 1.5-mL
Eppendorf tube for cell counting.
3. Centrifuge the cells at 500 Â g for 5 min at room temperature.
4. During centrifugation, perform cell counting using trypan blue
exclusion assay to determine the cell number and viability.
5. After centrifugation, discard the supernatant and resuspend the
pellet in 1 mL of culture medium.
6. Transfer the cells to a 25-cm
2 cell culture flask containing 5 mL
of culture medium, adjust the seeding density to 1 Â 10
5 viable
cells/mL for the K562 cell line and 4 Â 10
5 viable cells/mL for
the Raji cell line.
218
Nontaphat Thongsin and Methichit Wattanapanitch
The following section describes a general protocol that we use for
site-specific integration of the GFP gene in cell lines for tracking
functional immune cell killing (Fig. 1). The protocol includes
culture of the K562, Raji, and NK-92 cell lines, preparation of
individual plasmids containing sgRNA, Cas9 and GFP genes, transfection of the plasmids into the cell lines, enrichment of the GFP
+
cells by FACS and limiting dilution. After clonal expansion, the
engineered GFP
+ K562 or Raji cells can be used as target or
non-target cells for the NK-92 cell killing, respectively. This
approach facilitates immune cell killing assay since we can easily
track and directly evaluate the efficiency of immune cell cytotoxic
function.
3.1 Culture of K562
and Raji Cell Lines
3.1.1 Thawing
1. Warm K562 and Raji cell culture medium to room
temperature.
2. Quickly thaw the cryopreserved K562 and Raji cells in a 37
C
water bath until only a small ice crystal remains.
3. Wipe the outside of the cryovial with 70% ethanol.
4. Gently pipette the cell suspension and transfer to a 15-mL
conical tube containing 9 mL of RPMI-1640 basal medium.
5. Centrifuge the cells at 500 Â g for 5 min at room temperature.
6. Discard the supernatant and resuspend the pellet in 5 mL of
culture medium.
7. Transfer the cell suspension into a 25-cm
2 cell culture flask and
incubate at 37
C, 5% CO 2 (see Note 1).
3.1.2 Passaging
Passaging is performed when the cells reach a maximum density:
K562 cell line, 1 Â 10
6 viable cells/mL, and Raji cell line, 3 Â 10
6
viable cells/mL.
1. Gently resuspend the cells using a serological pipette and transfer the cell suspension to a 15-mL conical tube.
2. Aliquot a small amount of the cell suspension into a 1.5-mL
Eppendorf tube for cell counting.
3. Centrifuge the cells at 500 Â g for 5 min at room temperature.
4. During centrifugation, perform cell counting using trypan blue
exclusion assay to determine the cell number and viability.
5. After centrifugation, discard the supernatant and resuspend the
pellet in 1 mL of culture medium.
6. Transfer the cells to a 25-cm
2 cell culture flask containing 5 mL
of culture medium, adjust the seeding density to 1 Â 10
5 viable
cells/mL for the K562 cell line and 4 Â 10
5 viable cells/mL for
the Raji cell line.
218
Nontaphat Thongsin and Methichit Wattanapanitch