MOE430 GeneAtlas, GSE10246
Sternlicht, Mark
Dear Drs. Su, Walker, or whomever,
Though there are details pertaining to the preparation & stimulation of bone marrow & peritoneal macrophages in Lattin 08, I’ve been unable to locate details regarding such steps for other primary lines profiled in the MOE430 GeneAtlas. Is there any such supplemental information on-line? I’m especially interested in the preparation, characterization & activation of your mast cell, NK cell, osteoblast and osteoclast cultures, and any information in this regard would be greatly appreciated.
Thank you.
Sincerely,
Mark D. Sternlicht, Ph.D.
Senior Scientist
FibroGen, Inc.
409 Illinois Street
San Francisco, CA 94158 USA
E-mail: mster...@fibrogen.com
Direct: (415) 978-1496
Fax: (415) 978-1908
Main: (415) 978-1200
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John Walker
Dear Mark,
I’ll do my best. Submitters now are all over the world and not always easy to track down.
Mast cells, generated at GNF:
We use the following media to differentiate mBMMC's:
|
FINAL |
stock |
||||
|
Media |
RPMI |
1000 |
1X |
100% |
|
|
Pen/ Strep/fungizone |
10ml |
1X |
100mM |
||
|
L-Gln |
10ml |
1X |
100mM |
||
|
NEAA |
20ml |
2X |
100mM |
||
|
NA-pyruvate |
20ml |
2X |
100mM |
||
|
FBS |
100mls |
10% |
100% |
||
|
b-ME |
10ul |
1uM |
14.3M |
||
|
rmIl-3 |
10ng/ml |
10ug/ml |
added fresh |
||
|
rmSCF |
25ng/ml |
25ug/ml |
A summary of the culturing process is below:
-For the first two weeks after initiation, cultures are fed/ changed 2 times per week, discarding the adherent cells. Cells are plated 0.5-1.0e6 cells/ml in 10 or 15cm dishes (not flasks). The culture media used is 50% old media / 50% new media. rmIL-3 (10ng/ml) and SCF (25ng/ml) are added fresh at each change.
- After the initial two weeks, cells are fed / changed one time per week. Cells will begin to expand after about 1.5-2 weeks and are assayed by FACS for FcER1a & c-kit expression beginning at week 2. The cells generally take 3-5 weeks to become >95% FcE+ / c-kit+. Cultures are not used for experiments unless >95% double positive.
- the cells generally remain 100% viable for at least an additional 3-4 weeks.
- for stimulation, cells are washed with complete RMPI (minus cytokines) and starved of SCF overnight, but IL-3 is included in the stimulation media with IgE.
NK cells, generated at GNF: Isolated by Multenyi’s mouse NK kit. I have no further information. Submitter is long gone.
Osteoblasts and osteoclasts, also from the Hume lab:
Osteoblast cells were generated by sequential digestion of calvaria from neonatal 2 day old c57bl/6 mouse pups. Set 1 and 2 were isolated from 2 independed groups of 16 pups. The cells were then grown for 7 days in MEM with 10% FBS, P/S and L-Glx. From Day to 7 to 14 the cells were differentiated in BGJb media containing 10% FBS, P/S, L-Glx, Ascorbic acid and betaglycerol phosphate.
Osteoclasts were made by standard methods, ie. grown for 5 days in DMEM media supplemented with P/, 10% Heat inactivated Serum Supreme , Glx, CSF-1, ascorbic acid and 20ng/ml of recombinant soluble human RANKL. Media was replaced at day 3 and harvested in RLT containing BME on day 5.
I hope that helps.
John
From: Sternlicht, Mark [mailto:mster...@Fibrogen.com]
Sent: Tuesday, April 17, 2012 8:27 AM
To: John Walker; a...@gnf.org
Subject: MOE430 GeneAtlas, GSE10246
Dear Drs. Su and/or Walker,
Sternlicht, Mark
Dear John,
Thanks for the surprisingly rapid reply and for the info you were able to provide. Since these were the main cell types I was interested in, any further info (if it exists) is of lower priority, but might be useful for any future questions of this sort. Otherwise, I have to say bravo for a truly valuable resource.
Thanks again.
Best,
Mark
---------------------------------------------------------------------------
Mark D. Sternlicht, Ph.D.
Senior Scientist
FibroGen, Inc.
409 Illinois Street
San Francisco, CA 94158 USA
E-mail: mster...@fibrogen.com
Direct: (415) 978-1496
Fax: (415) 978-1908
Main: (415) 978-1200
P Before printing, think about the environment.
This transmission contains information intended for the exclusive use of the individual or entity to whom it is addressed and may contain information that is proprietary, privileged, confidential and/or exempt from disclosure under applicable law. If you are not the intended recipient (or an employee or agent responsible for delivering this transmission to the intended recipient), you are hereby notified that any copying, disclosure or distribution of this information may be subject to legal action, restriction, or sanction. If you have received this transmission in error, please notify us immediately. Thank you.