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Functional characterization of the ‘PBX interacting protein’ (HPIP) in normal and malignant human haematopoiesis.

von Dr. Pak

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[1.] Pak/Fragment 056 01 - Diskussion
Zuletzt bearbeitet: 2014-04-06 07:42:20 Hindemith
Ahmed 2007, Fragment, Gesichtet, Pak, SMWFragment, Schutzlevel sysop, Verschleierung

Typus
Verschleierung
Bearbeiter
SleepyHollow02
Gesichtet
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Untersuchte Arbeit:
Seite: 56, Zeilen: 1-14
Quelle: Ahmed 2007
Seite(n): 460, Zeilen: 3-18
[Wells were rinsed once with 1 ml PBS and added to tube. 1 ml Trypsin-EDTA was added to each well and incubated for 3 to 5 minutes] and examined for detached cells. Once the adherent cells are detached, the wells are washed with more PBS and the medium collected in the appropriated tube.

The wells are finally washed with 1 ml IMDM containing 2 % FBS and transferred to the specific tube. The tubes were centrifuged at 1200 rpm for 10 minutes and the supernatant was removed without disturbing the cell pellet. Approximately 200ìL of medium was left along with the cell pellet and vortexed. To this 3ml of Methocult (H4434) methylcellulose medium was added and vortexed again. Each tube (contents of one well) was plated individually into 2 different (1.5 ml/dish) 35 mm petri dish with 1 ml syringe (without needles attached). Different dishes (6-8) were placed in a 15 cm petri-dish along with an additional 60 mm open dish containing 5 ml sterile water to maintain humidity. The dishes are incubated at 37°C in humidified incubator (>95 %) with 5% CO2 in air for 16-20 days. Colonies were scored as positive if one or more BFU-E, CFU-GM or CFU-GEMM were detected or scored as negative if no colonies were present.

[The number of LTC-IC for the test cell population was calculated by dividing the total number of CFC detected in the culture by the average number of clonogenic progenitors per LTC-IC for the standard conditions used. Alternatively the values were expressed as LTC-IC derived CFC per number of test cells.115]


[115.Hogge DE, Lansdorp PM, Reid D, Gerhard B, Eaves CJ. Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor. Blood. 1996;88:3765-3773.]

Wells were rinsed once with 0.2 ml PBS and added to tube. 0.1 ml Trypsin-EDTA was added to each well and incubated for 3 to 5 minutes and examined for detached cells. Once the adherent cells are detached, the wells are washed with more PBS and the medium collected in the appropriated tube. The wells are finally washed with 0.2 ml IMDM containing 2% FBS and transferred to the appropriate tube. The tubes were centrifuged at 1200 rpm for 10 minutes and the supernatant removed without disturbing cell pellet. Approximately 0.1 ml of medium was left along with the cell pellet and vortexed. To this 1 ml of Methocult (H4435) methylcellulose medium was added and vortexed again. Each tube (contents of one well) was plated individually into 35 mm petri dish with 1 ml syringe (without needles attached). Several dishes (6-8) were placed in a 15 cm petri-dish along with an additional 60 mm open dish containing 5 ml sterile water to maintain humidity. The dishes are incubated at 37°C in humidified incubator (>95%) with 5% CO2 in air for 12 to 16 days. Colonies were counted and a well scored as positive if one or more BFU-E, CFU-GM or CFU-GEMM were detected or scored as negative if no colonies were present. [The LTC-IC frequency in the test cell population was calculated from the proportion of negative wells (no CFC present) and the method of maximum likelihood. Statistical analysis was performed using L-Calc™ software for limiting dilution analyses.]
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