Human Oncostatin M and Mouse LIF Support Mouse ES Cell Pluripotency &…

14 Апр 2015 | Author: | Комментарии к записи Human Oncostatin M and Mouse LIF Support Mouse ES Cell Pluripotency &… отключены
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The CJ7 ES cell line was a gift of Tom Gridley. ES cells maintained in high-glucose Dulbecco’s essential medium (DMEM) with 15% fetal bovine (FBS), 4 mM glutamine, 10 mM 2-mercaptoethanol, 1% MEM amino acids, 50 IU of penicillin per mL, 50 mg of per mL, and 103 U per mL LIF (ESGRO; Chemicon, Temecula CA) or 10 of oncostatin M (RD Systems, Catalog # ). Cells were grown on irradiated mouse embryonic cells (MEFs) or on 0.1% plates. Cells were every 2 days.

Embryoid Bodies (EBs)

bodies (EBs) were by plating 10 6 cells on a 6-well culture dish in DMEM with 10% FBS, 50 IU of penicillin per mL, 50 mg of per mL, and 4 mM glutamine. Cell clumps detached by flushing media the dish. The clumps were to a petri dish, and the media was daily.

For differentiation, EBs were in suspension for 4-6 days and then onto 0.1% gelatin 24-well plates and allowed to and grow for an additional 7 days to performing immunocytochemistry or extracting RNA for

RNA was extracted from undifferentiated ES and differentiated EBs using the RNAeasy kit according to manufacturer’s instructions. 500 ng of RNA was for reverse transcription with hexamers using the Superscript Synthesis System (Invitrogen) to manufacturer’s instructions. 2 mL cDNA was as a template for amplification.

All primers annealed at 55°C and amplified for 35

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All primary antibodies (rtxh/mOct3/4; gtxmNanog; mxβIII Tubulin; T; and gtxhHNF3β) used were RD Systems. Cells or EBs were onto coverslips in 24-well Cells were fixed in 4% for 20 minutes at room temperature and washed 4 times in PBS containing 1% BSA

Cells were blocked 10% donkey serum containing Triton® X-100 in PBA for 45 minutes. antibody was then added in at 10 μg/mL for 3 hours, after cells were washed 3 in PBA, 5 minutes each Cells were then in NorthernLights™ 557-conjugated secondary (RD Systems) at 5 mg/mL for 1 hour, washed as before.

Levels of activated STAT3 assessed with the Active DuoSet IC® Kit (RD Systems). Nuclear were made from ES cells grown either on or off according to kit instructions. Protein were quantitated by the Bradford

STAT3 activation levels determined according to manufacturer’s and then normalized to overall levels.

All procedures were by the University of Michigan Committee on Use and of Animals. Animal care was in accordance with the principles and outlined in the National Research Guide for the Care and Use of Laboratory After five serial on MEFs in media containing LIF or OSM, cells were and 16 ES cells were microinjected blastocysts.

Blastocysts were collected superovulated C57BL/6NCrl female (Charles River Laboratories, MA) mated with C57BL/6J X males (The Jackson Bar Harbor, ME). Microinjected were transferred to pseudopregnant females the day of injection using methods. Chimeras were to C57Bl/6J females (The Laboratory, Bar Harbor, ME) to test for transmission.

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