Hybridoma Cloning

Question 1

Poisson Distribution

Answer 1

Random distribution of cells in culture plate, so number of cells per well will follow Poisson distribution
Avg 1 cell/ well, 37% of the wells will contain 0 cells and 63% will contain one or more hybridoma
Growth in>63% = >1cell/well avg and colonies cannot be considered true clones

Question 2

Medium

Answer 2

Rich medium: IMDM-20 HAT + 20% FBS medium + 10% conditioned medium from myeloma parent cells
Supplies req growth factors (IL-2, IL-4, IL-13 and other cytokines)

Question 3

Timing

Answer 3

Hybridomas are unstable and have irregular # chromo

As they undergo rapid division, they could throw off chromo, losing ability to produce Ab

Question 4

Cloning assures:

Answer 4

All cells in cell line are producing Ab of interest
Monoclonality - should arise from a single cell
Allows selection of stably dividing cells secreting high amounts Ab

Question 5

Why do we use different concentrations of cells?

Answer 5

Ensure growth

Need to go higher than 1 cell/well to protect against dilution or calculation errors

Question 6

Why do we need to add growth supplements when growing at low concentration?

Answer 6

Cells require other cells in close proximity to get growth
Feeder cells (irradiated splenocytes ot thymocytes) or conditioned media used

Question 7

Factors affecting success of single cell cloning

Answer 7

Distrib of cells in medium (Poisson)

Cloning efficiency

Question 8

Expansion post-cloning

Answer 8

Can be injected into peritoneum where they will grow as tumors
Ascites/fluid can be harvested