3.7.2 Evaluation
of Cytotoxic Activity Using
Flow Cytometry Analysis
1. Determine the viability of the target cells using Zombie
Aqua™ Fixable Viability Kit in the GFP
+ population.
2. Killing capacity of the NK-92 cells can be calculated using the
following formula:
%Specific Lysis ¼
100 Â %Sample Lysis À Basal Lysis
ð
Þ
½
100 À %Basal Lysis
ð
Þ
The sample lysis represents the cell lysis in the presence of
effectors at a given effector to target ratio whereas the basal lysis
represents the cell lysis in the absence of effector cells.
4 Notes
1. After thawing, we recommend culturing the cells for at least
3 days for cell recovery and keep monitoring the cell density for
passaging.
2. The NK-92 cells tend to grow in aggregates and are sensitive to
overgrowth and medium exhaustion. Ensure that the cells are
cultured in the appropriate cell density with regular medium
change.
3. The amount of loaded plasmid for gel electrophoresis depends
on the concentration of the extracted plasmid, at least 10 ng of
the extracted plasmid should be used for qualification.
4. The percentage of agarose gel is dependent on the length of the
DNA fragment. We use 0.7% agarose gel for evaluation of
plasmid, genomic DNA, or large DNA fragment, and 1–1.5%
agarose gel for the DNA fragment smaller than 1000 bp.
5. It is important to choose the appropriate restriction enzyme for
plasmid digestion. We recommend using the restriction
enzyme(s) that can cut at least 2 sites with different fragment
lengths.
6. The reaction components and incubation time for plasmid
digestion depend on the restriction enzyme; the digestion
condition should follow the manufacturer’s instruction. In
this protocol, we use the FastDigest restriction enzyme
(Thermo Fisher Scientific).
7. The plasmids used for transfection should be of high quality as
poor quality of plasmid will decrease the transfection efficiency.
We recommend concentrating the plasmid using the ethanol
precipitation method. However, this step is not necessary if the
amount of loaded plasmid for nucleofection does not exceed
10% of the total volume.
8. Alternatively, 20-μL Nucleocuvette™ Strip can be used. In this
case, the number of the cells and the amount of plasmid should
CRISPR/Cas9-Mediated GFP Knock-in in K562 and Raji Cell Lines
227
of Cytotoxic Activity Using
Flow Cytometry Analysis
1. Determine the viability of the target cells using Zombie
Aqua™ Fixable Viability Kit in the GFP
+ population.
2. Killing capacity of the NK-92 cells can be calculated using the
following formula:
%Specific Lysis ¼
100 Â %Sample Lysis À Basal Lysis
ð
Þ
½
100 À %Basal Lysis
ð
Þ
The sample lysis represents the cell lysis in the presence of
effectors at a given effector to target ratio whereas the basal lysis
represents the cell lysis in the absence of effector cells.
4 Notes
1. After thawing, we recommend culturing the cells for at least
3 days for cell recovery and keep monitoring the cell density for
passaging.
2. The NK-92 cells tend to grow in aggregates and are sensitive to
overgrowth and medium exhaustion. Ensure that the cells are
cultured in the appropriate cell density with regular medium
change.
3. The amount of loaded plasmid for gel electrophoresis depends
on the concentration of the extracted plasmid, at least 10 ng of
the extracted plasmid should be used for qualification.
4. The percentage of agarose gel is dependent on the length of the
DNA fragment. We use 0.7% agarose gel for evaluation of
plasmid, genomic DNA, or large DNA fragment, and 1–1.5%
agarose gel for the DNA fragment smaller than 1000 bp.
5. It is important to choose the appropriate restriction enzyme for
plasmid digestion. We recommend using the restriction
enzyme(s) that can cut at least 2 sites with different fragment
lengths.
6. The reaction components and incubation time for plasmid
digestion depend on the restriction enzyme; the digestion
condition should follow the manufacturer’s instruction. In
this protocol, we use the FastDigest restriction enzyme
(Thermo Fisher Scientific).
7. The plasmids used for transfection should be of high quality as
poor quality of plasmid will decrease the transfection efficiency.
We recommend concentrating the plasmid using the ethanol
precipitation method. However, this step is not necessary if the
amount of loaded plasmid for nucleofection does not exceed
10% of the total volume.
8. Alternatively, 20-μL Nucleocuvette™ Strip can be used. In this
case, the number of the cells and the amount of plasmid should
CRISPR/Cas9-Mediated GFP Knock-in in K562 and Raji Cell Lines
227