SEB-1 cell line细胞株细胞系 BioVector NTCC质粒载体菌种细胞基因保藏中心

SEB-1 cell line细胞株细胞系 BioVector NTCC质粒载体菌种细胞基因保藏中心
SUBCULTURE: Human Adult Sebocytes
Adult sebocytes should be passaged for subculture when they are no more than 70-80%
confluent. Note that all cells are shipped at passage 3 after establishing a primary culture. For
primary sebocytes, we guarantee up to one passage.
1. Pre-warm complete SEB-1 medium, 0.25% trypsin/ 2.21mM EDTA solution and sterile
Phosphate Buffered Saline (PBS) Ca2+/Mg2+ free, in a water bath at 37C.
2. Aspirate medium and gently wash the cells 2-3 times with sterile PBS, to remove all traces of
medium.
3. Remove the PBS and add 1ml/T-25 flask of pre-warmed 0.25% trypsin/ 2.21mM EDTA solution.
4. Incubate the cells at 37C. Monitor cell detachment, under the microscope, after 2 minutes. Tap
the flask gently to loosen the cells. If the cells are still attached, place them at 37 C for another
1-3 minutes. Note: A longer incubation in trypsin can damage the sebocytes.
5. Neutralize the trypsin using an equal volume of 0.5 mg/ml soybean trypsin inhibitor. Collect all
the cells in a conical tube containing 2ml of SEB-1.
6. Note: If some of the cells remain attached after the recommended time, we suggest that you
neutralize the 0.25% trypsin/ 2.21mM EDTA solution by adding an equal volume of 0.5 mg/ml
soybean trypsin inhibitor, remove the solution (along with any detached cells) to a 15ml conical
tube and then add 2ml of complete SEB-1. To harvest the remaining attached sebocytes, wash
the plate with PBS, add another 1ml of trypsin to the flask and repeat step 4. Centrifuge to
obtain cell pellets as described below and combine resuspended cells prior to counting.



7. Centrifuge at 300xg, for 5 minutes at 20C.
8. Aspirate the medium, and gently resuspend the cell pellet in a desired volume of SEB-1 and
proceed to cell counting.
9. Seed cells at 10,000-20,000 cells/cm2, (0.25-0.5x106 cells per T25 flask) in 6 ml of SEB-1. If
using 48 or 96 well plates seed the cells at 20,000-40,000 cells/cm2. Ensure cells are evenly
suspended when plating large numbers of plates or flasks. Do not agitate plates and flasks



after plating. Place in a humidified incubator at 37C and 5% CO2, making sure the surface is
level for even cell distribution.
10. Replace the medium 24 hours after plating and then every 2-3 days until they are 70-80%.
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