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Ectopic expression of the homeobox gene Cdx2 is the key transforming event in a mouse model of t(12;13)(p13;q12) acute myeloid leukemia; a novel mechanism in AML

von Dr. Vpr

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[1.] Vpr/Fragment 034 01 - Diskussion
Zuletzt bearbeitet: 2014-03-17 12:34:39 Graf Isolan
Fontanari Krause 2006, Fragment, Gesichtet, SMWFragment, Schutzlevel sysop, Verschleierung, Vpr

Typus
Verschleierung
Bearbeiter
SleepyHollow02
Gesichtet
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Untersuchte Arbeit:
Seite: 34, Zeilen: 1-17
Quelle: Fontanari Krause 2006
Seite(n): 92, Zeilen: 2-17
[The] cells were subjected to half-media change every 7 days and their proliferation assessed on the same day by counting viable cells after trypan exclusion. To generate IL-3 dependent cell lines, successfully transduced bone marrow cells were cultured directly after sorting in DMEM, 15% FBS with mIL-3 alone (6ng/ml). A half-media change was done every 7 days.

3.6 Colony Forming Cells Assay (CFC-assay).

To check the differentiation and clonogenic potential of the BM cells expressing the various genes mentioned above, we performed a CFC assay by testing colony formation in methylcellulose. The CFC–assay was performed by culturing highly purified transduced cells (500 /dish) in 35mm-diameter Petri dishes directly after sorting in 1ml methylcellulose supplemented with cytokines (Methocult M3434), and incubated at 370C in humidified CO2 incubator. Colonies were counted microscopically on 7 to 9 days after plating according to standard criteria (Schwaller et. al., 1998). Re-plating capacity of clonogenic progenitors was assayed by re-plating the primary colonies on secondary methylcellulose dishes. Again colonies were counted day at 7 to 9 after the secondary re-plating.

The cells were subjected to half-media change every 7 days and their proliferation assessed on the day of media change by counting viable cells after trypan exclusion. To generate IL-3 dependent cell lines, successfully transduced bone marrow cells were cultured directly after sorting in DMEM, 15% FBS with IL-3 alone (6 ng/ml). A half-media change was done every 7 days.

5.10.4.2. Colony Forming Cells Assay (CFC-assay)

To analyze the differentiation and clonogenic potential of the transduced BM cells, we performed CFC assays by testing colony formation in methylcellulose. The CFC–assay was done by culture highly purified transduced cells (500/dish) in 35mm-diameter Petri dishes directly after sorting in 1ml methylcellulose supplemented with cytokines (Methocult M3434). Colonies were counted microscopically on 7 to 9 days after plating according to standard criteria (Schwaller, 1998). Re-plating capacity of clonogenic progenitors was assayed by replating the primary colonies (500 cells/dish) on secondary methylcellulose dishes. Again colonies were counted at 7 to 9 days after the secondary re-plating.

Anmerkungen

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Sichter
(SleepyHollow02) Schumann


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