Differentiation of 3T3-L1 Cells: Difference between revisions
| Line 19: | Line 19: | ||
*Cells can be grown at 37C + 8% CO2, and split 10-20X. Typically healthy fibroblasts will repopulate in 2-3 days after a 10X dilution. | *Cells can be grown at 37C + 8% CO2, and split 10-20X. Typically healthy fibroblasts will repopulate in 2-3 days after a 10X dilution. | ||
*Split cells when at about 80% confluence. Confluence initiates the differentiation program, so if cells reach confluence, you will need to thaw another DMSO stock and start over. Splitting cells will normally not reverse this | *Split cells when at about 80% confluence. Confluence initiates the differentiation program, so if cells reach confluence, you will need to thaw another DMSO stock and start over. Splitting cells will normally not reverse this | ||
*When splitting cells, wash cells 2x with sterile PBS then add 0.05% trypsin and sit at room temperature for 2-5 min. Be careful not to over-trypsinize cells | *When splitting cells, wash cells 2x with sterile PBS (-/-) then add 0.05% trypsin and sit at room temperature for 2-5 min. Be careful not to over-trypsinize cells | ||
*Cells can normally be passaged up to about passage # 25 without problems. | *Cells can normally be passaged up to about passage # 25 without problems. | ||
*Plate cells out in whichever format you require your adipocytes in (i.e. 12 well plates or 15 cm dishes) | *Plate cells out in whichever format you require your adipocytes in (i.e. 12 well plates or 15 cm dishes) | ||