Experimental design Investigate the effect of either sera or

Experimental design: Investigate the effect of either sera or substrates on the proliferation of 3T3 fibroblasts.

1. Provide step-by-step written protocol. The protocol should include the seeding conditions (cell densities, culture vessels, medium composition, type of serum or substrate tested, culturing period investigated, etc) and the proper controls.

-Specifically, you will want to test their effect on cell proliferation, so:

a. imagine how you may set up this experiment, and

b. propose how you will determine whether an effect occurred or not (read-out).

Solution

Condioned Medium (CM) is used for the proliferation of 3T3 fibroblasts. Fresh DME-FBS will be added to ensure the culture. After 24 hours of incubation, the medium will be removed and fresh DME-FBS will be added. Again incubate this medium at 37C for 24 hours. After incubation, centrifuge at 1470g for 10 minutes. The supernatant will be collected and it will be used as fresh condioned medium (CM) for 3T3 fibroblasts. Serum Free condioned medium and serum Free unconditioned medium is also prepared simultaneously. SME will not be added in serum Free unconditioned medium and it can be supplement with target testing cells. The viability of the cells treated with CM and UCM is determined by attaching of cells in the plastic surface. After removal of medium, it can be treated with trypan blue for 24 hours. After removing of staining solution, the attached cells are counted using microscope.