什么是流式细胞术?
Flow cytometry is a method for the measurement of relative size, 粒度, and fluorescence of single cells in a population of cells suspended in a liquid medium. Samples are stained with specific fluorescently-labeled reagents (e.g.抗体). The labeled cells are illuminated by a 488 or 635 nm laser, and the fluorochrome emission (fluorescent intensity) and light scatter (cell size and complexity) are recorded.
流式细胞仪
The UIC has a 正欲 FACSCalibur Flow Cytometer. 这台流式细胞仪(2000年6月). 日期)是一个四色, dual-laser, bench-top system capable of cell analysis using forward scatter, 边撒, and detection of fluorescence in four distinct color regions: > 670 nm (deep red), 653-669 nm(红色), 564-606 nm(橙色), 515-545 nm(绿色). The instrument has a Mac G4 host computer, and most instrument functions are computer controlled. The instrument also has two lasers for exciting fluorochromes: an argon laser, 哪种能发出488纳米的蓝宝石光, 红色二极管激光器发射635纳米的光.
What fluorochromes are commonly used with this instrument?
Fluorochromes commonly used with these lasers include fluorescein, 噻唑橙, 吖啶橙, 藻红蛋白, propidium碘化, allophycocyanin, pharmingen/BD's 藻红蛋白-Cy5 (Cy-Chrome) and peridinin chlorophyll protein (PerCP). Please note that UV and green excited dyes such as DAPI and rhodamine and its derivatives cannot be used with this cytometer.
我应该和UIC的谁谈谈流式细胞术?
请致电603-862-0632或发电子邮件给马克·汤利 mtownley@cisunix.主要研究.edu 了解更多信息.
我如何提交样品进行分析?
你需要预约马克·汤利. Please make an appointment at least 24 hours BEFORE you'd like your samples run. 如果你迟到或错过约会, you will still be billed for the entire reserved time, 除非你提前至少24小时取消.
We currently do not train anyone outside of the UIC on the instrument, 所以所有的样本都是由UIC的技术人员操作的. All samples to be run should be non-toxic, non-infectious, non-pathogenic, and fixed. The UIC reserves the right to refuse to run a sample if there is a concern about the toxicity and/or safety of the sample. For more on the 澳门葡京网赌游戏's flow cytometry policy, please refer to the University's Biological and Chemical Safety Plan.
A 提交表格样本 should be filled out for all samples prior to submission.
什么大小的细胞可以分析? 浓度是多少??
The FACSCalibur Flow Cytometer is best suited for the analysis of aqueous suspensions of cells or particles with diameters between 1 and 50 um (microns). It is important that you filter aggregated samples with 70 um nylon cell strainer (made by Falcon, part number 352350) before running them on the instrument. If cells have aggregated to the point that you can see them in the sample tube (and a vortex mixer does not break up the clumps), 样品将堵塞仪器上的流动电池.
Samples should ideally contain 1 x 106 cells or particles per mL.
Samples should be submitted in capped 12 x 75 mm Falcon brand 5 mL polystyrene round bottom tubes (part number 352003).
我需要多少样品?
Even though sample consumption can be varied between 12 uL/min and 60 uL/min (allowing analysis of relatively small samples), 请提交至少1mL的体积.
还有什么我要考虑的吗?
To make conclusions about your experiment, the proper controls are critical. 确保你有积极和消极的控制. You will always end up needing one more control than you have; so when in doubt, prepare more controls then you think you'll ever need. A negative control is an unstained sample (autofluorescence!!) or a sample stained only with the secondary, if this is the system you are using. If you have samples stained with more than one fluorochrome per test tube, bring in plenty of sample stained with each fluorochrome individually. 这是为了补偿. For an explanation of this procedure, see Mario Roederer's website on 补偿.
The UIC staff will provide as much technical support, advice, and direction as possible. 然而, the UIC is not responsible for problems caused by poor sample preparation or flawed experimental design.
有用的网站和链接
- 流式细胞仪设备 在康涅狄格大学
- 普渡大学细胞术实验室
- 国际分析细胞学学会 直接督导下
- 流式细胞术在网络上
流式细胞术的好书
- Flow Cytometry: First Principles by Alice Givan (really good introduction)
- Howard Shapiro的实用流式细胞术
- Flow Cytometry: A Practical Approach by Michael Ormerod (good information on DNA)
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