The O9-1 mouse cranial neural crest cell line was purchased from MilliporeSigma (SCC049) and cultured in complete ES cell medium containing 15% FBS and LIF (MilliporeSigma, ES-101-B) with 100 units/ml penicillin-streptomycin (Invitrogen) at 37°C in a humidified atmosphere containing 5% CO2 according to the manufacturer’s instructions. For osteogenic or chondrogenic differentiation, O9-1 cells were cultured with StemMACS OsteoDiff Media or StemMACS ChondroDiff Media (Milteny Biotec), respectively. Osteogenic differentiation and chondrogenic differentiation were evaluated by alizarin red or alcian blue staining as previously described [70 (link),73 (link)]. To knockdown Tmem2 expression in O9-1 cells, we used lentivirus-mediated shRNA transduction. Lentivirus particles expressing an shRNA that is validated to deplete mouse Tmem2 (Mission shRNA, TRC Clone ID: TRCN0000295501, MilliporeSigma) and control lentivirus particles expressing an shRNA that does not target any known genes (Mission shRNA, MilliporeSigma SHC005) were purchased from MilliporeSigma. Lentivirus particles were added to O9-1 cells cultured in growth media supplemented with 5 μg/ml polybrene and cultured for 2 days. Cells transduced with lentiviral shRNAs were selected and maintained in the presence of 10 μg/mL puromycin.
Es 101 b
Manufactured by Thermo Fisher Scientific
The ES-101-B is a laboratory equipment designed for general use. It serves as a platform for various experimental and analytical tasks. The device's core function is to provide a controlled environment for such applications.
Automatically generated - may contain errors
2 protocols using es 101 b
1
Cranial neural crest cell line differentiation and knockdown
2
Knockdown of Tmem2 in O9-1 mouse neural crest cells
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The O9-1 mouse cranial neural crest cell line was purchased from MilliporeSigma (SCC049) and cultured in complete ES cell medium containing 15% FBS and LIF (MilliporeSigma, ES-101-B) with 100 units/ml penicillin-streptomycin (Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO 2 according to the manufacturer's instructions. To knockdown Tmem2 expression in O9-1 cells, we used lentivirus-mediated shRNA transduction. Lentivirus particles targeting mouse Tmem2 (Mission shRNA, TRCN0000295501) and their controls (Mission shRNA, SHC005) were purchased from MilliporeSigma. Lentivirus particles were added to O9-1 cells cultured in growth media supplemented with 5 μg/ml polybrene and cultured for 2 days.
Cells transduced with lentiviral shRNAs were selected and maintained in the presence of 10 μg/mL puromycin.
Cells transduced with lentiviral shRNAs were selected and maintained in the presence of 10 μg/mL puromycin.
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