集落形成细胞（CFC）的含量对人体的造血细胞
Summary
集落形成细胞（CFC）的检测是在体外实验中，造血祖细胞形成半固体培养基中的菌落。结合菌落形态，细胞形态，流式细胞仪是用来评估的祖细胞的增殖和分化沿不同的造血谱系能力。
Abstract
人造血干/祖细胞通常从骨髓，脐带血，外周血和用于研究造血和白血病。他们有能力分化成淋巴和髓细胞谱系。集落形成细胞（CFC）的测定是用来研究的增殖和分化的造血祖细胞的能力，在半固体培养基中形成的殖民地模式。一个固定数量的输入细胞形成的菌落的数量和形态提供初步信息有关的祖细胞能够分化和增殖。从单个菌落或从整个板块收获细胞可以进一步评估它们的数量和分化状态，用流式细胞仪检测和形态学评估姬姆萨染色幻灯片。这种分析是有用的评估髓细胞分化，但没有淋巴。在这方面的长期髓系在其更广泛的意义上使用，包括粒细胞，单核细胞，红系，巨核细胞谱系。
我们已经用这种检测评估的首要人权从外周血CD34 +细胞衍生的细胞分化癌基因的影响。这样做的目的细胞转要么控制逆转录病毒构建或利益表达的原癌基因的构建，在这种情况下，NUP98 – HOXA9。我们采用一种常用的逆转录病毒载体MSCV – IRES – GFP，表达了产生的利益和GFP标记基因的双顺反子mRNA的。细胞被预先激活，越来越多的前两天，以逆转录病毒转导的细胞因子的存在。另外两天后，GFP +细胞是孤立的荧光激活细胞分选（FACS），并含有一个甲基纤维素半固体与细胞因子的补充和培养至菌落出现在表面上，一般为14天的中期混合。记录的数量和形态的殖民地。细胞，然后从板块中删除，洗涤，计数，和流式细胞仪检测和形态学检查。流式细胞仪检测表达在造血细胞表面标记的特异性抗体提供有关血统和成熟阶段的信息。 Wright – Giemsa染色后显微镜下单个细胞的形态学研究提供进一步的信息方面的血统和成熟。控制空载体转导细胞比较，揭示造血分化癌基因的影响癌基因转导。
Protocol
1。试剂的制备准备无菌FMS相关酪氨酸激酶3的1000倍的股票解决方案（FLT – 3）配体，粒细胞/巨噬细胞集落刺激因子（GM – CSF的），干细胞因子（SCF），血小板生成素（TPO），白细胞介素（IL）-3 ，IL – 6和根据制造商的指示。准备的无菌Retronectin股票溶液（1mg/ml的）也可根据制造商的说明。分成小等份，这些股票的解决方案，并保持在-20 ° C，避免反复冻融。 准备2％FBS的IMDM。 准…
Discussion
已广泛用于氟氯化碳检测，以确定造血祖细胞的增殖和分化的模式，并研究原癌基因的影响（4，5）。它利用液体培养的克隆实验，殖民地代表一个单系祖的后代，并作进一步的分析可以单独删除，。的CFC检测的限制，这是不足够更不成熟的祖细胞或造血干细胞的检测，发现这种细胞的长期培养启动细胞（LTC – IC）的测定（6，7）。这里所描述的实验表明，反洗钱相关基因NUP98 – HOXA9抑制髓系和红系…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢华盛顿大学医学院和巴恩斯犹太医院在圣路易斯，密苏里州的阿尔文J. Siteman癌症中心，为使用高速细胞分选的核心，提供流式细胞仪检测设备和排序服务。这项工作是由国家资助卫生研究院R01 HL082549和K02 HL084179（NRY）的支持。 Siteman癌症中心是支持部分由NCI癌症中心支援津贴P30 CA91842。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| IMDM | Life Technologies | 12440 | ||
| FBS | StemCell Technologies | 06150 | ||
| L-Glutamine | Life Technologies | 25030 | ||
| Penicillin/Streptomycin (PS) | Life Technologies | 15140 | ||
| FLT-3 ligand | Peprotech | 300-19 | ||
| GM-CSF | Peprotech | 300-03 | ||
| SCF | Peprotech | 300-07 | ||
| TPO | Peprotech | 300-18 | ||
| IL3 | Peprotech | 200-03 | ||
| IL6 | Peprotech | 200-06 | ||
| Bovine Serum Albumin | Sigma- Aldrich | A7030 | ||
| EDTA | Fisher Scientific | BP118 | ||
| Retronectin | Takara Bio Inc | T100A | ||
| HBSS | Life Technologies | 14175 | ||
| PBS | Life Technologies | 14200 | ||
| Trypan Blue solution (0.4%) | Sigma-Aldrich | T8154 | ||
| Methocult GF+ H4435 | StemCell Technologies | 04445 | ||
| Human Methylcellulose Enriched Media | R & D Systems | HSC005 | ||
| Wright/ Giemsa stain | Harleco | 64571 | ||
| Phosphate Buffer Solution, pH 6.4 – Giordano formula | Ricca Chemical Company | 1450 | ||
| Methanol | Fisher Scientific | A412-4 | ||
| Cytoseal 60 | Thermo Scientific | 8310 | ||
| Normal Mouse Serum | Rockland Immunochemicals | D208 | ||
| Anti-Human CD11b phycoerythrin-conjugated | BD Biosciences | 555388 | ||
| Anti-Human CD33 allophycocyanin-conjugated | BD Biosciences | 551378 | ||
| Anti-Human CD45 phycoerythrin-Cy7-congjugated | BD Biosciences | 557748 | ||
| Anti-Human CD71 phycoerythrin-conjugated | BD Biosciences | 555537 | ||
| Anti-Human CD235a allophycocyanin-conjugated | BD Biosciences | 551336 | ||
| Anti-Human CD45 phycoerythrin-Cy7-congjugated | BD Biosciences | 557748 | ||
| 24-well non-treated tissue culture plates | BD Biosciences | 35-1147 | ||
| 30 mm non-treated dish | StemCell Technologies | 27150 | ||
| 100 mm tissue culture dish | Fisher Scientific | 08-757-12 | ||
| Gridded scoring dishes | StemCell Technologies | 27500 | ||
| 15 ml centrifuge tubes | BD Biosciences | 35-2097 | ||
| 50 ml centrifuge tubes | BD Biosciences | 35-2070 | ||
| Syringes 3 ml | StemCell Technologies | 28240 | ||
| 16 gauge blunt-end, 1½ inch needle | StemCell Technologies | 28110 | ||
| 50 μm CellTrics cell filter | Partec | 04-004-2327 | ||
| Hemocytometer | Fisher Scientific | 0267110 | ||
| TPX sample chambers | Thermo Scientific | A78710018 | ||
| Fisherbrand Superfrost/Plus Microscope Slides, Precleaned | Fisher Scientific | 12-550-15 | ||
| Shandon filter cards | Thermo Scientific | 5991022 | ||
| Shandon cytospin slide holder | Thermo Scientific | 59920063 | ||
| Shandon Complete Staining Assembly 100 | Thermo Scientific | 100 | ||
| Kimwipes | Kimberly-Clark Corporation | 34155 | ||
| 1 μm filter paper | VWR | 28307-134 | ||
| Inverted microscope | Nikon | Diaphot | ||
| Microscope camera | Nikon | DS-F11 | ||
| Microscope | Olympus | BX51 | ||
| Microscope camera | Olympus | DP71 | ||
| Scanner | Microtek | Scanmaker 4 | ||
| Vortex mixer | Fisher Scientific | 12-812 | ||
| Tissue culture incubator | Sanyo | MCO-18AIC | ||
| Cytospin | Shandon | Cytospin 2 | ||
| Bench-top centrifuge | Eppendorf | 5810-R | ||
| Water purification system | Barnstead | Nanopure-Diamond |
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Tags
Colony Forming Cell (CFC) AssayHuman Hematopoietic CellsBone MarrowCord BloodPeripheral BloodHematopoiesisLeukemogenesisLymphoid LineageMyeloid LineageSemisolid MediumProliferationDifferentiation PatternColoniesInput CellsFlow CytometryGiemsa-stained SlidesMyeloid DifferentiationOncogenesPrimary Human CD34+ CellsRetroviral ConstructNUP98-HOXA9MSCV-IRES-GFP
Cite This Article
Sarma, N. J., Takeda, A., Yaseen, N. R. Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells. J. Vis. Exp. (46), e2195, doi:10.3791/2195 (2010).