"MEFs and HeLa cells were maintained in DMEM-GlutaMAX (Thermo Fisher Scientific) containing 4.5 g l −1 of glucose supplemented with 1 mM sodium pyruvate (Gibco), 100 μM non-essential amino acids (Gibco) and 10% FBS (Biochrom). IMR90 cells were maintained in MEM-GlutaMAX (Thermo Fisher Scientific) and 10% FBS. MEF and HeLa cell lines were maintained at 37 °C under 5% CO 2 and ambient oxygen. IMR90 cells were maintained at 37 °C under 5% CO 2 and 3% O 2. Cell lines were routinely tested for Mycoplasma infection."
"Cell numbers were monitored by Trypan Blue exclusion and cell counting using the Countess automated cell counter (Thermo Fisher Scientific). Cells were seeded at equal densities and grown to confluency over a period of 72 h without medium changes unless stated otherwise. Opti-MEM + GlutaMAX (Gibco) and lipofectamine RNAiMax (Invitrogen) were used for reverse transfection of endoribonuclease-prepared short interfering RNA (esiRNA) and short interfering RNA (siRNA) for 72 h. A list of the esiRNAs and siRNAs used in this study is provided in Supplementary Table 1."
MEFs and HeLa cells were cultured in DMEM-GlutaMAX with 4.5 g/l glucose, 1 mM sodium pyruvate, 100 μM non-essential amino acids, and 10% FBS. IMR90 cells were cultured in MEM-GlutaMAX with 10% FBS. MEF and HeLa lines were maintained at 37 °C with 5% CO2 and ambient oxygen; IMR90 cells at 37 °C with 5% CO2 and 3% O2. Cells were tested for Mycoplasma and counted by Trypan Blue exclusion using an automated counter. Reverse transfection used Opti-MEM + GlutaMAX and Lipofectamine RNAiMax to deliver esiRNA and siRNA for 72 h. Deoxyribonucleoside supplementation during senescence used 100 μM of each deoxynucleoside refreshed every 72 h. Immortalized WT and Yme1l−/− MEFs were produced via SV40 large T antigen immortalization, Cre recombinase transduction, clonal expansion, and genotyping.
Read at Nature
Unable to calculate read time
Collection
[
|
...
]