In this class, you will prepare your 3T3 fibroblasts for cryopreservation, freez

Question

In this class, you will prepare your 3T3 fibroblasts for cryopreservation, freeze them, move them to liquid nitrogen storage, thaw and re-plate them, and examine them for recovery efficiency. You will need to write a protocol that you can follow when cryopreserving your cells.  Illustrate your procedure and write a detailed protocol for both cryopreservation and thawing safely.  Submit your illustration and protocol to Blackboard. Use the following sources of information as you prepare your procedures:

Your lecture notes.

Textbook reading: Chapter 15 (pp. 307-320).

Protocols "Freezing and Thawing 3T3 Fibroblasts" and "Cryopreservation and Thawing" posted on Blackboard.

Explanation / Answer

Freezing

combine cells fro each 10 cm dish and pellet (spin for 2-3 min at 1000 rrpm.

Resuspend the pellet in 1ml of complete media + 10% DMSO 1ml per each 10 cm dish.

Aliquot 1ml of resuspend cells/vial and freeze slowly in isopropanol @~80 then store in the liquid nitrogen freezer.

thawing

thaw one tube of cells quickly at 37for 2mins.

Rinse 70% ethanol

Add cells to 10-20ml of preconditioned complete growth media

Once cells sit down do a 100% media change into complete growth media to remove DMSO and any dead cells

Once cells have reached a pre confluent state usually 1-2 days after thawing cells can be split to carry for differentiation.

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