After you have determined the cleavage efficiency of the pooled cell population, isolate single cell clones for further validation and banking. You can isolate single cell clones from the selected pool using limiting dilution cloning (LDC) in 96‑well plates or by single cell sorting using a flow cytometer.
Example single cell sorting procedure in a 96-well plate using flow cytometer
You can sort single cells per well into a 96-well plate format using a flow cytometer with single cell sorting capability. After sorting and expanding the single cell clones, analyze and characterize the clonal populations using suitable assays. The following is an example single-cell sorting procedure with 293FT cells.
- Wash the transfected 293FT cells in each well of the 24-well plate with 500 μL of PBS. Carefully aspirate the PBS and discard.
- Add 500 μL of TrypLE cell dissociation reagent to the cells and incubate for 2–5 minutes at 37°C.
- Add 500 μL of complete growth medium to the cells to neutralize the dissociation reagent. Pipette the cells up and down several times to break up the cell aggregates. Make sure that the cells are well separated and are not clumped together.
- Centrifuge the cells at 300 × g for 5 minutes to pellet.
- Aspirate the supernatant, then wash the cell pellet once with 500 μL of PBS.
- Resuspend 1 × 106 cells in 1 mL of FACS buffer, then add propidium iodide (PI) to the cells at a final concentration of 1 μg/mL. Keep the resuspended cells on ice.
- Filter the cells using suitable filters before analyzing them on a flow cytometer with single cell sorting capability.
- Sort PI-negative cells into a 96-well plate containing 100 μL of complete growth medium. If desired, you can use 1X antibiotics with the complete growth medium.
- Incubate the plates in a 37°C, 5% CO2 incubator.
- Scan the plates for single cell colonies as soon as small aggregates of cells are visible under a 4X microscope. Colonies should be large enough to see as soon as 7–14 days (usually after first week, depending on the growth rate of the cell line). You can perform image analysis to ensure that the colonies are derived from single cells.
- After image analysis, continue incubating the plates for an additional 2–3 weeks to expand the clonal populations for further analysis and characterization.
Characterize edited clones
You can analyze the single cell clones for purity and the desired genotype (homozygous or heterozygous allele) by various molecular biology methods such as genotyping PCR, qPCR, next-generation sequencing, or western blotting.