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Typus
KomplettPlagiat
Bearbeiter
SleepyHollow02
Gesichtet
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Untersuchte Arbeit:
Seite: 53, Zeilen: 5-29
Quelle: Ahmed 2007
Seite(n): 42-43, Zeilen: 10ff.;1ff.
3.2.11. Colony forming cell assay

Haematopoietic colony-forming cells (CFC) were assayed using methylcellulose-based medium (MethoCult H 4434) (fig. 3.2.11). Required number of pre-aliquoted tubes of MethoCult medium was thawed overnight under refrigeration (2-8°C) or at room temperature. The cells were diluted with IMDM + 2% FBS to 10X the final concentration(s) required for plating. For a duplicate assay 0.3 ml of diluted cells was added to 3 ml MethoCult tube and the contents vortexed vigorously. After about 5 minutes, 1.1 ml of cell: methylcellulose mixture was dispensed into 35 mm culture dishes using sterile a 3 ml syringe and16-gauge bunt-end needle. The 35 mm culture dishes were placed into 10 cm petri dish along with an extra 35 mm dish containing sterile water to maintain humidity and placed in a CO2 incubator at 37°C and >95% humidity.

CFC numbers were evaluated after an incubation period of 12-14 days and distinguished into following classes: Colony-forming unit-erythroid (CFU-E): Produces 1-2 cell clusters containing a total of 8-200 erythroblasts. A CFU-E consists of mature erythroid progenitors that require erythropoietin (EPO) for differentiation. Burst-forming unit-erythroid (BFU-E): Produces a colony containing >200 erythroblasts in a single or multiple clusters. A BFU-E consists of more immature progenitors than CFU-E and requires EPO and cytokines with burst-promoting activity such as Interleukin-3 (IL-3) and Stem Cell Factor (SCF) for optimal colony growth.

Colony-forming unit-granulocyte, macrophage (CFU-GM): Produces a colony containing at least 20 granulocyte cells (CFU-G), macrophages (CFU-M) or cells of both lineages (CFU-GM). CFU-GM colonies arising from primitive progenitors may contain thousands of cells in single or multiple clusters. Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM):

3.2.8 Human CFC Assay

Haematopoietic colony-forming cells (CFC) were assayed using methylcellulose-based medium (MethoCult; H 4434) (fig 3.3). Required number of pre-aliquoted tubes of MethoCult medium was thawed overnight under refrigeration (2-8°C) or at room temperature. The cells were diluted with IMDM + 2% FBS to 10X the final concentration(s) required for plating. For a duplicate assay 0.3 ml of diluted cells was added to 3 ml MethoCult tube and the contents vortexed vigorously. After about 5 minutes, 1.1 ml of cell: methylcellulose mixture was dispensed into 35 mm culture dishes using sterile a 3 ml syringe and16-gauge bunt-end needle. The 35 mm culture dishes were placed into 10 cm petri dish along with an extra 35 mm dish containing sterile water to maintain humidity and placed in a CO2 incubator at 37°C and >95%

[Page 43]

humidity. CFC numbers were evaluated after an incubation period of 12-14 days and distinguished into following classes:

Colony-forming unit-erythroid (CFU-E): Produces 1-2 cell clusters containing a total of 8-200 erythroblasts. A CFU-E consists of mature erythroid progenitors that require erythropoietin (EPO) for differentiation.

Burst-forming unit-erythroid (BFU-E): Produces a colony containing >200 erythroblasts in a single or multiple clusters. A BFU-E consists of more immature progenitors than CFU-E and require [sic] EPO and cytokines with burst-promoting activity such as Interleukin-3 (IL-3) and Stem Cell Factor (SCF) for optimal colony growth.

Colony-forming unit-granulocyte, macrophage (CFU-GM): Produces a colony containing at least 20 granulocyte cells (CFU-G), macrophages (CFU-M) or cells of both lineages (CFU-GM). CFU-GM colonies arising from primitive progenitors may contain thousands of cells in single or multiple clusters.

Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM):

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