Opti mem glutamax medium

Female individuals were used at the age of 8 to 12 weeks. Progenitor cells were derived from bone marrow of the mice strains mentioned above. The progenitor cells were retrovirally transduced with estrogen-regulated Hoxb8 and selected for 4 weeks in the presence of stem cell factor (SCF) to generate neutrophil progenitor cell lines [25 (link)]. Polyclonal progenitor cell lines were cultured in Opti-MEM + GlutaMAX medium (Life Technologies, Darmstadt, Germany) supplemented with 10% FCS, 30 μM ß-mercapthoethanol, 1 μM ß-estradiol (Sigma-Aldrich, Taufkirchen, Germany) and 1% supernatant from SCF-producing CHO cells. The SCF producing cell line was kindly provided by Hans Häcker (St. Jude Children’s Research Hospital, Memphis, TN, USA). Differentiation was induced by ß-estradiol removal.

Female mice were used at the age of 8–12 weeks. Progenitor cells were derived from murine bone marrow. The progenitor cells were retrovirally transduced with estrogen-regulated Hoxb8 and selected for 4 weeks in the presence of stem cell factor (SCF) to generate neutrophil progenitor cell lines (Wang et al., 2006 (link)). Polyclonal progenitor cell lines were cultured in Opti-MEM + GlutaMAX medium (Life Technologies, Darmstadt, Germany) supplemented with 10% FCS, 30 µM ß-mercaptoethanol, 1 µM ß-estradiol (Sigma-Aldrich, Taufkirchen, Germany) and 1% supernatant from SCF-producing CHO cells. The SCF producing cell line was kindly provided by Hans Häcker (St. Jude Children’s Research Hospital, Memphis, TN, USA). Differentiation was induced by ß-estradiol removal. The differentiation status of the cells was controlled for each experiment microscopically by Giemsa staining and was similar for WT and gene-deficient Hoxb8 neutrophils. MyD88^-/- TRIF^-/- Hoxb8 neutrophils were a gift from Hans Häcker.

Post-transcriptional gene silencing was performed by the reverse transfection method. Cells were seeded at a density of 1.75 × 10⁴ in a T25-cm² flask on the day of transfection. To allow complex formation, 700 µL of Opti-MEM GlutaMax medium (Invitrogen, Carlsbad, CA, USA) without antibiotics were placed in a sterile tube with 10 µM siRNA stock solution (Life Technologies, Carlsbad, CA, USA) and TransIT-X2 (Mirus Bio LLC, Madison, WI, USA) in a 1:1 ratio. Tubes were incubated for 20 min on ice and finally transferred into the flasks. Silencer select^® siRNAs (SIGMAR1, s20086 and TMEM97, s26204, Life Technologies, Carlsbad, CA, USA) and validated Negative Universal ControlTM (Invitrogen, Carlsbad, CA, USA) final concentrations ranged from 40 nM to 80 nM. Cells were incubated for 24 to 96 h with the complexes and assayed for knockdown of target gene expression.

60,000 FACS-sorted fetal LSK cells from iMLL-ENL and wild type mice were cultured in Opti-MEM + GlutaMax medium (Gibco) supplemented with 10% fetal calf serum (FCS; HyClone), 1% penicillin/streptomycin (Gibco) and 0.02% 50 mM 2-mercaptoethanol (Gibco) and 25 ng/mL stem cell factor (SCF), 25 ng/mL Flt3-ligand (Flt3l), 20 ng/mL interleukin (IL) −7, 10 ng/mL IL-6, 10 ng/mL granulocyte (G) -colony stimulating factor (CSF), 10 ng/mL granulocyte-macrophage (GM)-CSF, 10 ng/mL IL-3 and 0.1% doxycycline (to both iMLL-ENL and wild-type cells). After two days, cells were harvested, washed 3x with HBSS and 500,000 cells/replicate were collected for proteomic analysis and stored in 10% TCA at −80 °C until sample preparation.

The immortalized mouse neuronal astrocyte cell line C8D1A [Astrocyte type I clone] (ATCC^® CRL2541^TM) was purchased from ATCC and was cultured in Dulbecco's modified Eagle's medium GlutaMAX medium (Gibco, 10569-010) containing 10% fetal bovine serum (Sigma, F1051) and 1% penicillin/streptomycin in a 5% CO₂ atmosphere at 37 °C. The cells were infected overnight with a 1% brain homogenate from terminally sick mice infected with mouse-adapted scrapie prion strains (22L, RML, and ME7). The mouse neuroblastoma cell line N2a was cultured in Opti-MEM GlutaMAX medium (Gibco, 51985-034) containing 10% fetal bovine serum (Sigma, F1051) and 1% penicillin/streptomycin in a 5% CO₂ atmosphere at 37 °C. CAD5 cells are a central nervous system catecholaminergic cell line (77 (link)) and were cultured in Opti-MEM GlutaMAX medium containing 10% bovine growth serum (Hyclone, SH30541.03), and 1% penicillin/streptomycin in a 5% CO₂ atmosphere at 37 °C. Immortalized MEFs were cultured in Dulbecco's modified Eagle's medium GlutaMAX medium (Gibco, 10569-010) containing 10% fetal bovine serum (Sigma, F1051) and 1% penicillin/streptomycin in a 5% CO₂ atmosphere at 37 °C.