Does anyone have a full protocol for collecting and lysing differentiated adipocytes (3T3-LI cells)?

Lanisha LaTice Patterson @Lanisha-Patterson

19 September 2023 0 5K Report

I have been trying to conduct an experiment looking at what conditioned media from a specific type of cancer cell does to differentiated adipocytes. After treating the cells, I collect them, sonicate them, spin them down to remove the fat layer, and then lyse them using RIPA buffer with PIC inhibitor. After lysing the remaining cell pellet I am getting little to no protein. Does anyone have a protocol for the collection and lysing of differentiated 3T3-LI cells? Any suggestions on how many cells need to be seeded to get enough protein lysate? Centrifuge speeds and time? Would adding SDS or Triton X-100 to the lysis buffer assist in extracting lipolytic proteins? i appreciate any advice here.

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