流式细胞术中应⽤的荧光染料介绍
当我们在进⾏多⾊流式分析的时候，对分析成功的⼀个关键影响因素是每个抗体选择何种荧光染料标记。通常会有很多可⾏的结合，我们在做选择的时候有很多的因素需要我们去考虑。对任何单克隆抗体，其阴性和阳性的信噪⽐相差四到六倍都取决于荧光素的使⽤。相对的荧光强度也取决于使⽤的仪器。⾼密度表达的抗原我们可以应⽤任何荧光素标记的抗体来检测，低密度表达的抗原就需要我们应⽤⾼信噪⽐⾼的荧光素，譬如PE或者APC标记的抗体来充分检测分离出阳性表达的细胞与阴性表达的细胞。学校食堂自查报告
⾃发荧光
个别的细胞有着他们各⾃特异性⽔平的⾃发荧光（荧光信号产⽣于他们⾃⾝）。当在全波长荧光通道中进⾏观察的时候，⾃发荧光信号在长波长（>600nm）明显的降低。
对于有较⾼⽔平⾃发荧光类型的细胞，如长期组织培养的细胞，应使⽤较⾼发射波长的荧光染料（APC、APC-Cy7等）标记抗体，通常他们会有⼀个较好信噪⽐的结果。
对于那些不是有较多⾃发荧光类型的细胞，如新鲜的细胞，可以使⽤FITC 标记的抗体了。
以下为各种常⽤荧光素介绍：
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Alexa Fluor 488 has a spectrum almost identical to that of fluorescein isothiocyanate (FITC), but with extraordinary photostability. Becau of this photostability, it has become a choice for fluorescent microscopy applications and has become popular in cytometry applications. It is detected in the FL1 detector of the FACSCalibur or FACScan. Unlike other fluorochromes with similar emission spectra, Alexa Fluor 488 is pH innsitive over a broad range.
Alexa Fluor 633 is a practical alternative to APC as well as Cy5. Alexa Fluor 633 conjugates can be ud in multi-color flow cytometry with instruments equipped with a cond red lar or red diode. It is detected in the FL4 detector of the FACSCalibur. The FACScan cannot be ud to detect Alexa Fluor 633 conjugates becau the FACScan lacks a red lar or diode. Like other Alexa Fluor dyes, Alexa Fluor 633 exhibits uncommon photostability, making it an ideal choice for fluorescent microscopy.
A great number of different Alexa Fluor dyes exist that are beyond the scope of this introductory fluorophore ction. Many manufacturers ll directly-conjugated Alexa Fluor antibodies. Know that Molecular Probes' Zenon Antibody Labeling Kits, which are available for all of their Alexa Fluor dyes, make it possible to rapidly and quantitatively label antibodies from a purified antibody fraction or from a crude antibody preparation such as rum, ascites fluid or a hybridoma supernatant. See ww
数学四年级下/rvlets/publications?id=150 for specifics.
Allophycocyanin (APC) is an accessory photosynthetic pigment found in blue-green algae. APC has 6 phycocyanobilin chromophores per molecule, which are similar in structure to phycoerythrobilin, the chromophore in phycoerythrin or PE. APC tandem dyes, APC-Cy5.5 and APC-Cy7, are also available. APC has a 650-nanometer wavelength absorption maximum and a 660-nanometer fluorescence emission maximum. APC can be ud in flow cytometers equipped with dual lars for multi-color analysis. Like Alexa Fluor 633, APC is excited using the helium-neon red diode lar (633 nanometers) of the FACSCalibur and is detected on the FL4 detector. APC cannot be detected on the FACScan as that instrument is not equipped with a red lar.
APC-Cy7 is a tandem conjugate system that combines APC and a cyanine dye (Cy7) and has an absorption maximum at
~650 nanometers. This tandem us the efficiency of the fluorescence light energy transfer between the two fluorochromes. When excited by light from a helium-neon lar, the excited fluorochrome (APC) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength. The resulting fluorescent emission maximum is in the deep red at approximately
767 nanometers. APC-Cy7 run on a FACSCalibur results in dim expression becau the FL4 detector's optical filter is centered for APC emission (660 nanometers) and not the longer red wavelengths excited with the helium-neon diode. It is recommended that special precautions be taken with this conjugate, and cells stained with them, to protect the fluorochrome from long-term exposure to visible light.
Carboxyfluorescein Diacetate (CFSE) can be ud to track asynchronous cell division. Cell division results in quential halving of the initial fluorescence, resulting in a cellular fluorescence histogram.
The peaks labeled 1, 2, 3, 4 and 5 reprent successive generations of CFSE-cultured cells.
Cy3 and Cy5 are excited by the 488-nanometer line of an argon lar and the 633-nanometer line of a helium-neon diode or lar, respectively. The conjugates can be ud in flow cytometry but typically do not give the fluorescence intensity comparable to that of PE or APC. Applications where a smaller dye is required are more appropriate for the dyes. The fluorochromes are well suited for fluorescent microscopy.
邢台云梦山Enhanced Cyan Fluorescent Protein (eCFP) cannot be analyzed using the FACScan or FACSCalibur as this molecule requires excitation in the violet range. The protein is easily detected u
sing the violet lar of the LSRII, or the violet lines of the MoFlo's argon or krypton lars. This molecule has an excitation maximum at 475 nanometers and is best excited using the MoFlo's 457nm line of the argon or argon-krypton mixed gas lar. U of the 407nm violet lar of the LSRII will result in weak eCFP expression. More detailed discussion of this molecule can be found in the next ction of this web site, the ction on Fluorescent Proteins.
人居环境整治工作总结Enhanced Green Fluorescent Protein (eGFP) can be excited at 488 nanometers with a peak emission at 509 nanometers and is detected in the FL1 detector on the FACSCalibur or FACScan. The MoFlo and LSRII are able to distinguish between concurrently expressing eGFP and eYFP cells if the proper optical filters and experimental controls exist. More detailed discussion of this molecule can be found in the next ction of this web site, the ction on Fluorescent Proteins. Enhanced Yellow Fluorescent Protein (eYFP), a yellow-shifted variant of the eGFP molecule, is also excited at 488 nanometers with a peak emission at 535 nanometers and is also detected in the FL1 detector on the FACSCalibur or FACScan. More detailed discussion of this molecule can be found in the next ction of this web site, the ction on Fluorescent Proteins.
Fluorescein isothiocyanate (FITC) is currently the most commonly ud fluorescent dye for flow cytometry analysis. When excited at 488 nanometers, FITC has a green emission that's usually colle
cted at 530 nanometers, the FL1 detector of the FACSCalibur or FACScan. FITC has a high quantum yield (efficiency of energy transfer from absorption to emission fluorescence) and approximately half of the absorbed photons are emitted as fluorescent light. For fluorescent microscopy applications, FITC is ldom ud as it photobleaches rather quickly though in flow cytometry applications, its photobleaching effects are not obrved due to a very brief interaction at the lar intercept. FITC is highly nsitive to pH extremes.
Peridinin chlorophyll protein (PerCP) has a 677 nanometer maximum emission, red, when excited at 488 nanometers and is detected on the FL3 detector of the FACSCalibur or FACScan. A PerCP tandem dye is also available (PerCP-Cy5.5). PerCP is not suited for the high-powered lasing (>150mW) applications, such as on the MoFlo, due to its photobleaching characteristics. PerCP conjugates can only be obtained from Becton Dickinson and its subsidiary, Pharmingen. Phycoerythrin (PE or R-PE) has a huge absorption coefficient and almost perfect quantum efficiency. In vivo, it functions to transfer light energy to chlorophyll during photosynthesis. It is one of the brightest dyes ud today and emits in the
yellow/orange at about 570 nanometers. Tho accustomed to fluorescent microscopy may not be familiar with this fluorochrome as it photobleaches rather quickly under a microscope.
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Phycoerythrin-Cy5 (PE-Cy5) is a tandem conjugate where PE is coupled to the cyan dye, Cy5. When excited by 488-nanometer light, the excited fluorochrome (PE) is able to transfer its fluorescent energy to the cyanine molecule, which then fluoresces at a longer wavelength in the red at 670 nanometers. This tandem dye is known by a confusing myriad of names to include Becton Dickinson's CyChrome, Caltag and Sigma's Tri-Color, GIBCO's RED670, Coulter's PC5, and probably others. Other PE conjugates exist, e.g., PE-Cy5.5 and PE-Cy7, that will not be discusd in this introductory fluorophore ction. It is recommended that special precautions be taken with this conjugate, and cells stained with them, to protect the fluorochrome from long-term exposure to visible light.
Phycoerythrin-Texas Red (PE-Texas Red) is a tandem conjugate where PE is coupled to Texas Red. Similar to other tandem conjuates, when excited by 488-nanometer light, the excited fluorochrome (PE) is able to transfer its fluorescent energy to the Texas Red molecule, which then fluoresces at a longer wavelength with a peak in the orange at 612 nanometers. This tandem is also known by other names such as Red 612 and ECD (Electron Coupled Dye). Know that PE-Texas Red conjugates run on the FACScan or FACSCalibur will result in dull expression due to the extant optical filters. This is not obrved on the LSRII or MoFlo when equipped with the appropriate optical filters for this conjug
ate. There is considerable overlap of emission when running PE and PE-Texas Red specimens.
Propidium Iodide (PI) is a membrane-impermeant dye that stains by nondiscriminately intercalating into every 4th or 5th nucleic acid ba pair, binding both DNA and RNA. Once bound, PI undergoes a conformational change and becomes ~40 times brighter. Propidium iodide has a broad emission spectrum with a peak in the orange at 620 nanometers. A number of assays employ propidium iodide. Cells or nuclei with altered DNA content are identified by staining alcohol-fixed, RNA-treated cells or nuclei with this dye (e below).
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青少年英文The first peak (M1) on the left reprents normal diploid cells. The next major peak (M2) reprents tumor cells with incread DNA content and DNA index of 1.27. Propidium iodide can be combined with additional fluorescent antibodies that are specific for a unique cell population and allow for more accurate S pha analysis of multiple overlapping populations.
Propidium iodide has also been employed for many years as a marker for viability as the disrupted membranes of dead cells allow the dye to pass freely to the nucleic acids. However, this dye is very sticky; it will stick to sample tubing and, given sufficient time exposure to living cells, living cells will appear to be propidium iodide positive. Given this dye's broad emission spectrum and its sticky prop
erties, contemporary flow cytometry labs have replaced propidium iodide with other nucleic acid dyes, e.g., 7-AAD (listed below), TO-PRO-3, or DAPI, among many others, for viability measurements though propidium iodide remains the most commonly ud dye for DNA content analysis.
Texas Red has an excitation maximum in the yellow-orange range of the color spectrum; conquently, Texas Red cannot be excited on any of the benchtop analyzers in the core facility. It can, however, be detected using one of green lar lines of the MoFlo's argon lar but ideally using the yellow lar line of the MoFlo's krypton lar. When excited, Texas Red has an excitation maximum in the orange at 612 nanometers.
7-Aminoactinomycin D (7-AAD), like propidium iodide, is a DNA intercalating dye but 7-AAD is specific for C-G ba pairs. It is well suited for viability measurements and also for apoptosis experiments where it's paired with Annexin V. Unlike propidium iodide, this dye has minimal emission bleed from the FL3 detector into the FL2 (PE) detector on the FACSCalibur or FACScan. Whereas PI can be detected in either FL2 or FL3, though it is typically detected in FL2, of the FACScan or FACSCalibur, 7-AAD is detected in FL3.
PE：Phycoerythrin 藻红蛋⽩；488nm，575nm（橙红）
FITC，异硫氰酸荧光素 488nm，525nm（绿
APC：英⽂全名：allophycocyanin，中⽂名：别藻青蛋⽩，最⼤吸收峰：650nm，最⼤发射荧光峰：660nm，适⽤于双激光流式仪，可被600-640nm波长的激光激发。
PerCP英⽂全名：peridinin chlorophyll protein，中⽂名：多甲藻叶绿素蛋⽩，最⼤激发波峰490nm，被488nm的氩离⼦激光激发后，发射光峰值约为677nm，与FITC、PE进⾏多⾊荧光染⾊补偿重叠较少，⾮特异性结合也较少。虽然较易使⽤，但量⼦产量不⾼，多⽤于检测表达较⾼的抗原。
