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about culturing megakaryocytes

stemcells

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#1 faith2010

faith2010

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Posted 21 November 2012 - 02:24 AM

Hii all..
I want to ask your experience about how culturing megakaryocytes.
So few weeks ago, I tried to culture mouse bone marrow cells for CFC assay (I grew in MethoCult separately and also MegaCult-Stem cells technologies). After several days, I can see, count, and picked up colonies from MethoCult. But for megacult culture, It was a bit tough for me to pick up the colonies for next cuturing. and after around almost 2 weeks culturing in the medium, looks like more adherent cells (like fibroblast) are more growing.

so, at first I culture Bone marrow cells with this MegaCult:

Megacult-C Medium without cytokine (Stemcell Technologies, 04950)
50 ng/ml rhThrombopoietin
20 ng/ml rhIL-6
10 ng/ml rmIL-3
- Collagen (Stemcell Technologies, 04902 ; 3mg/ml): I use 0,6mL in every 1 mL MegaCult
Cells number was: 1.0×105 cells/dish

then, after around 8 days, I picked up the colonies, and start culturing in this medium:

IMDM : 16 ml
15% FBS : 3 ml
2 mM L-Glu (200 mM) : 200 µl
100 µM 2ME (100 mM) : 20 µl
1% BSA (20%) : 1 ml
10 µg/ml insulin (5 µg/ml) : 40 µl
200 µg/ml transferrin (100 mg/ml) : 40 µl
50 ng/ml rh TPO (50 µg/ml) : 20 µl
20 ng/ml rh IL-6 (20 µg/ml) : 20 µl
50 ng/ml rh IL-11 (50 µg/ml) : 20 µl
10 ng/ml rm IL-3 (10 µg/ml) : 20 µl

If you have any experiences how to pick up the megakaryocytes more easily (because it was too sticky, and looks like they like to grow in the sticky area), could you please give any suggestion how to prepare MegaCult medium before adding the Bone marrow cells?

Thank you all

Faith





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