Cloning limiting dilution method

The methods leading to the isolation of fused cells are fairly standard. Two different methods are used for cloning (i) limiting dilution or, (ii) cloning on semi-solid or in viscous media. Once the desired... 

Initial growth, screening, and cloning by limiting dilution The most popular method for the production of clones, whenever the fraction of specific antibody-secreting hybrids is lower than 10 is by limiting dilution. 

Fig. 1 Methods to study delayed drug hypersensitivity reactions in humans, (a) T cells - mainly from the peripheral blood, but also from tissue sections (skin, kidney), proliferate when exposed to drugs in cell culture. The proliferating cells can be expanded and then cloned by limiting dilutions, and functional and phenotypic analysis performed to evaluate how drugs are stimulatory for T cells and which functions they perform, (b) Immunhistology of skin (and kidney) using antibodies to cell surface and cytokines, etc. the example shows that in a maculopapular exanthema both CD4+ and CD8+ T cells also express perforin...

All procedures are done in a laminar flow hood using aerosol barrier pipette tips and sterile technique. Methods used to transform B. hermsii by electroporation are similar to those procedures previously described for B. burgdorferi (15,21, 22,23). Spirochetes were cultivated and cloned by limiting dilution in liquid mBSK-c. 

Cloning by limiting dilution in 96-well plates is the most efficient method. A feeder cell or conditioned medium (Protocol 5) must be used. 

---