フローサイトメトリー (FCM) / FACS テクノロジーセンター

フローサイトメトリーの図

1. Air pressure is used to drive cells through a FACS machine.
2. 'Sheath fluid,' basically isotonic buffered water, dilutes the cell suspension inside the machine.
3. The cells, present as a suspension in an isotonic buffer, ultimately move past a laser and electronic detector system in a single file.
4. Different FACS machines may be equipped with lasers that emit lights of different wavelengths.
5. As the cells move past the laser, they reflect and refract the light from the lasers, and they also glow or fluoresce, depending on the nature of fluoresce on or in the cells.
6. Various detectors pick up such signals from the cells. These 'signals' are also referred to as 'parameters'.
7. The FSC or forward scatter parameter indicates the light reflected back by a cell. In general, it is a measure of the cell size Larger cells have bigger FSC values, while cell debris and dying cells (generally speaking) have low FSC values.
8. The SSC or side scatter parameter indicates the light refracted by the cell at an angle to the incident laser light Cells that are more granular a are dying (generally speaking) have higher SSC values.
9. A cells, besides reflecting light, will fluoresce if it has fluorochromes that are excitable by light of particular wavelengths emitted by the lasers. The wavelength of this fluorescent light is always higher than that of the incident laser light It varies depending on the nature of the fluorochrome.
10. For example. FITC fluorochrome fluoresces green while PE fluorochrome fluoresces orange.
11. FACS machines have different detectors for such different 'colors.' Thus, a cell's FL1, FL2, FL3, etc, parameters are measured.
12. Each sample analysis thus involves the measurement of all these parameters for a specified number of cells (usually, 10,000) The data recorded look something like this:

Cell FSC SSC FL1 FL2 FL3
1211 101 211 253 890 567
1212 103 291 353 890 767

13. In the end, a software application is used to plot the values as histograms, dot-plots, etc.
14. Fluorochromes do not emit light of just one wavelength It is instead a range of wavelengths Similarly, the electronic detectors do not detect light of just one wavelength, but a range of wavelengths Thus, light may 'leak' from and get detected by more than one detector.
15. TNis is important to consider and 'compensate' for if one is detecting multiple fluorochromes on or in the same cell.
16. Fluorochromes in a cell can be of various types For example, a cell might be expressing a green fluorescent protein such as GFP. A cell's DNA might be stained with a fluorochrome such as propidium iodide. Or. antibodies, either with fluorochrome chemical tags or 'tagged' with 'secondary antibodies' with such tags, might be bound to antigen-bearing molecules in or on the cell.

フローサイトメトリーは細胞表面および細胞内分子の発現を解析し、異種細胞集団における異なる細胞の種類を特徴づけ、定義し、単離された亜集団の純度を評価し、細胞のサイズおよび細胞容積を分析するために、広く使用されている方法で、単細胞の同時マルチパラメーター解析が可能です。

これは主に、タンパク質を検出する蛍光標識抗体、またはDNAに結合するヨウ化プロピジウムなどの特定の細胞関連分子に結合するリガンドによって生成される蛍光強度を測定するために使用されます。

染色手順は、細胞培養物または組織サンプルから単細胞懸濁液を作製することを含みます。次いで、細胞を、未標識または蛍光色素標識抗体を有するチューブまたはマイクロタイタープレート中でインキュベートし、フローサイトメーターで解析します。