Hybridoma cloning
Summary
Hybridoma cloning is mainly used to (1) clone antibody-positive holes. (2) Separating antibody-secreting cells, antibody-nonsecreting cells, desired antibody (specific antibody) secreting cells, and other unrelated antibody-secreting cells.
Operation method
finite dilution
Principle
Hybridoma cloning generally refers to the cloning of antibody-positive wells. This is because hybridoma cloning after HAT screening does not guarantee that there is only one clone in a well. In practice, there may be several or even more clones, which may include antibody-secreting cells, antibody-non-secreting cells, desired antibody (specific antibody) secreting cells, and other unrelated antibody-secreting cells. Clonization is required to separate these cells from each other. The principle of clonization is to clone hybrid clones that test positive for antibodies as early as possible, otherwise antibody-secreting cells will be suppressed by antibody-nonsecreting cells because antibody-nonsecreting cells grow faster than antibody-secreting cells, and competition between the two results in the loss of antibody-secreting cells. Even cloned hybridoma cells need to be recloned periodically to prevent mutation or chromosome loss in hybridoma cells, which would result in the loss of antibody-producing ability.
Materials and Instruments
Hybridoma Cells Move 1. Prepare feeder cell layer (homocyte fusion) 1 day before cloning. 2. Blow the hybridoma cells to be cloned gently dry from the culture well and count. 3. Adjust the cells to 3--10 cells/ml. 4. Take the cell culture plate of the feeder cell layer prepared on the first day and add 100μl of diluted cells to each well. Incubate in 37℃, 5% CO2 incubator . 5. Change the solution on day 7 and every 2-3 days thereafter. 6. 8-9 days visible cell clone formation, timely detection of antibody activity. 7. Transfer the cells from the positive wells to 24-well plates to expand the culture. 8. Each clone should be frozen as soon as possible. Caveat Each clone should be frozen as soon as possible. There are many methods of clonization, the most commonly used being the limited dilution method and the soft agar plate method. Common Problems There are many methods of clonization, the most commonly used being the limited dilution method and the soft agar plate method. For more product details, please visit Aladdin Scientific website.
Feeding solution
Petri plates, CO2 incubator