Greetings!
 
Single-cell cloning has gained increasing importance as CRISPR/Cas9 genome editing technique has entered routine laboratory practice. However, the success of positive single-cell cloning is technically challenging. The long growing time and the extremely low efficiency of obtaining a positive single-cell clone are the major challenges. The rate of single-cell clone formation may be affected by many factors; two of which are particularly important, one is the survival rate of inoculated cells, and the other is the cell proliferation ability.
 
1 How to improve the survival rate of inoculated cells?


1) Maintain a stable pH
 
Most of the cell culture media are formulated with CO2 carbonate buffer which is suitable for the partial pressure of CO2 in incubators. However, when this buffer is in atmospheric conditions, carbon dioxide will evaporate from the medium, which will cause the pH of the medium to rise to the alkaline range (in the medium containing phenol red, the color will become more purple). This will significantly affect the viability of cells. Therefore, the HEPES buffer system can be used instead of the CO2 carbonate system. It is a better and safer choice.
 
2) Keep everything at the right temperature
 
Before digesting and plating cells to 96-well plate, the culture medium, trypsin and PBS should be warmed up to 37 ℃, to keep cells under a relatively stable condition.
 
3) For some sensitive or fragile cell lines, the number of inoculated cells can be increased correspondingly, for example, from 50 cells per 96-well plate to 80 cells per plate.
 
2 How to improve the proliferation of cells?
 
1) Passage appropriately
 
Cells were inoculated with limit dilution and cultured to form microcolonies (∼ 50-100 cells). Transfer a microcolony to a fresh well of a 96-well plate. When the cells in the new well grow to 80% confluence, the cells are transferred to a well of a 48-well plate. Allow the cells to proliferate until a sufficient number of cells can be harvested for validation.
 
2) Adding supplements
 
The universal factors that are required in nearly all cell lines, which have been identified in the literature include insulin, transferrin, and selenium. For certain cell lines, Ethanolamine may also be critical and, in some cases, attachment factors such as fibronectin, laminin, vitronectin, and growth factors may be beneficial.
 
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