• Protocol for Limiting Dilutions

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NOTE: The sooner after cell fusion you can do a limiting dilution, the better your chances of retaining strong positive hybridomas.

1. Remove spleen cells from 1 or 2 mice, wash as for cell fusion (above), and suspend the cells in ca. 100 ml DMEM complete media containing HT.

2. Dilute hybridoma cells by 1x104 to 1x105. For normal cell cultures, this can be accomplished by placing 0.1 ml of hybridoma cells into 10 ml of washing media. Mix, and remove 0.1 ml to a tube containing 10 ml of spleen cell media (1 x 104 dilution). For very thickly growing cells, make two dilutions into washing media (1 x 106 dilution).

3. Plate 36 wells of a 96 well tissue culture plate with 0.2 ml of this mixture (7.2 ml) (GOAL: 5 hybridoma cells/well). This will leave 2.8 ml of the original cell suspension.

4. To the remaining 2.8 ml suspension add 7.2 ml of spleen cell suspension, and seed 36 wells with 0.2 ml of this mixture (GOAL: 1 hybridoma cell/well).

5. Finally, add 2.0 ml of spleen cell suspension and seed 0.2 ml into the remaining 24 cells (GOAL: 0.5 ml hybridoma cell/well).

The following steps are the original instructions:

1. Plate 36 wells of a 96 well tissue culture plate with 0.1 ml of this mixture (3.6 ml) (GOAL: 5 hybridoma cells/well). This will leave 1.4 ml of the original cell suspension.

2. To the remaining 1.4 ml suspension add 3.6 ml of spleen cell suspension , and seed 36 wells with 0.1 ml of this mixture (3.6 ml; 1.4 ml remaining) (GOAL: 1 hybridoma cell/well).

3. Finally, add 1.0 ml of spleen cell suspension and seed 0.1 ml into the remaining 24 cells (GOAL: 0.5 ml hybridoma cell/well).)

4. Depending of culture conditions, cloning efficiencies, counting, dilution error, 37% of the wells should have no growth, as determined by the Poisson distribution.

5. Test the wells that appear to be monoclonal for antibody production (ELISA). Freeze strong positive wells (as given below), and expand the cell line in vitro using a large tissue culture flask or in vivo by initiation of ascites fluid in mice (BALB/c).