Single-cell dilution

One usually tries to adjust experimental conditions so that only one plasmid or phage is introduced into a single cell. When that cell replicates on a plate of nutrient agar, it will produce millions of cells until a colony becomes visible to the naked eye. If cells are sufficiently diluted before they are spread onto the plate, each colony will consist of a clone derived from a single ancestral cell. If each cell originally contained only one copy of the phage or plasmid vector, each colony will contain recombinant DNA that is homogeneous. That is, each colony will contain cloned DNA. 

Allogeneic mixed thymocyte medium (4) is a conditioned medium used to aid cells recover from the fusion process. It also encourages division of hybridoma cells after dilution cloning when colonies derived from single cells are required. Rat thymi are used for this purpose to minimise the number of animals required to produce adequate numbers of cells. The medium contains a number of helper cytokines produced by the thymocytes. It is important that the thymocytes used are derived from two different rat strains as this causes co-stimulation and enhanced cytokine secretion by the cells. Mixed thymocyte medium must always be diluted with RPMI 1640 medium containing 15% FBS and is usually used at a dilution of 10-15%. 

These methods are, however, largely unnecessary when it is desired to clone continuous cell lines. Thus a dilute suspension of HeLa cells (containing about 100 cells in 5 ml) will grow up to form colonies, each derived from a single cell. It is important to prevent the movement of these cells (and hence mixing of clones) and sometimes they are overlayered with soft agar. 

Continue to the end of the tray to give a set of 2- fold, serial dilutions of the original suspension. An 8-place dispenser is invaluable for this operation. Several of the wells in row 7 or 8 should contain a single cell and this can be checked microscopically. 

Once a properly diluted and a single cell suspension of viable cells at the correct level of confluence is obtained, there is yet another parameter that should be controlled. Suspensions should be maintained in polypropylene containers rather than polystyrene. Typically, tumor cells adhere better to the latter and the number of cells being injected can change as cells adhere to the walls. 

Lohse AW, Dinkelmann M, Kimmig M, Herkel J, Meyer zum Buschenfelde KH (1996) Estimation of the frequency of self-reactive T cells in health and inflammatory diseases by limiting dilution analysis and single cell cloning. J Autoimmun 9 667-675. 

Generally, capillaries with inner diameters of 5-25 pm are used to inject and analyze single cells in CE. With such small capillaries, several features can be achieved, including low risk of injection of more than one cell at a time, low dilution factors after cell lysis, high resolving power, and rapidity at high electric field strengths. It is important to keep a very small dilution factor, as the... 

---