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View additional product information for CellROX™ Deep Red Flow Cytometry Assay Kit - FAQs (C10491)
9 product FAQs found
许多对哺乳动物细胞使用的染料也可以用于细菌。例如，CellROX Deep Red试剂经证实可以用于检测B. subtilis的活性氧（见参考文献http://www-brs.ub.ruhr-uni-bochum.de/netahtml/HSS/Diss/RaatschenNadja/diss.pdf）。如果您对某一特定染料有兴趣但不知道它对您的细菌是否有效，最佳途径是通过文献检索的方式查看它是否已经过试验。如果没有，则可能需要您亲自测试。
•钙流：每一种Oregon Green钙指示剂都可通过更高的亲和力结合胞内钙离子，提供适合很多应用的灵敏度范围。Oregon Green探针在Ca2+静息水平下发射绿色荧光；Ca2+浓度增加时，荧光强度会增加14倍。细胞通透配方（货号O6807）能加入到细胞培养基中并与流式细胞仪兼容。
•基于罗丹明的钙离子指示剂包含了大量不同的探针，用于探测Ca2+浓度的大小变化。该指示剂与钙离子结合后，会发出50倍增强的荧光。这一类波长范围的荧光可与GFP或绿色荧光染料结合，用于多重检测应用。Rhod-2, AM（货号R1245MP）专门靶向线粒体，可与流式细胞仪联用。
•膜电位：细胞凋亡初期的典型特征是线粒体紊乱，伴随着膜和氧化还原电位变化。我们提供一系列专门用于流式细胞术活细胞线粒体膜电位分析的产品，可最大限度避免影响细胞功能。对于细胞凋亡过程出现的线粒体膜电位损失，MitoProbe系列线粒体染色剂（货号M34150、M34151和M34152）可提供快速、简单和可靠的流式细胞术检测方法。MitoTracker染料（货号M7510和M7512）是用于染色活细胞内的线粒体的膜电位依赖型探针。在之后的流式细胞免疫化学、DNA末端标记，原位杂交或复染色步骤中，MitoTracker染料的染色图案全程保留。相比于只依赖线粒体膜电位的检测方法，线粒体通透性转移孔检测体系（货号M34153）可直接测定通透性转移孔开合情况。线粒体通透性转移孔（MPTP）是一个由线粒体内膜和外膜成分构成的非特异性通道。在细胞死亡过程中，此通道显现，参与线粒体成分释放。
•吞噬作用：在吞噬过程中，细胞内吞微粒（如微生物）。此过程对于免疫应答非常重要，同时对清除凋亡细胞也非常重要。研究吞噬作用的探针包括BioParticles指示剂——用荧光标记的细菌和酵母。
•使用淬灭/洗涤检测法来追踪吞噬过程能够表征简单的摄入，或利用一种pH指示剂监视吞噬途径的各个阶段。我们提供pHrodo Red或Green（货号A10010、P35361、P35364、P35365、P35366和P35367）标记的免洗检测体系和全血的免洗检测体系（货号A10025、A10026、P35381和P35382），都适用于流式细胞仪。
•pH改变：可使用荧光强度或比率计测定法测定生理学范围内的细微pH变化。pHrodo 染料（货号P35373和P35372）提供了pH2-9之间的信号强度调制，同时可以选择各种荧光波长。荧光右旋糖酐内吞示踪是分析细胞区室pH变化的常用方法。pHrodo染料的右旋糖酐偶联物（货号P35368和P10361）可用于区分从早期的核内体到溶酶体在内的各种囊泡，不需要洗涤和淬灭，是最完整的解决方案。
•活性氧：处于环境压力下的细胞通常含有超高水平的活性氧（ROS）。CellROX试剂是为检测和定量活细胞中的ROS而开发的荧光探针。这些细胞通透性试剂在还原态时不发荧光或发微弱的荧光；在被氧化后，就发出明亮的荧光且依旧位于细胞内。我们提供已通过流式细胞术验证的CellROX Green（货号C10492）CellROX Orange（货号C10493）和CellROX Deep Red（货号C10491）检测试剂盒。
首先，确保您同时拥有一个阴性（未处理）和一个阳性（ROS诱导）样本进行比较。100 µM甲萘醌1小时或是50 µM奈法唑酮24小时可以作为很好的阳性对照。H2O2 虽然不能很好地与CellROX染料兼容，但也可以用于阳性对照。某些如H2DCFDA的染料需要酯酶切割，所以不能在血清（血清中包含的酯酶可过早切割染料）存在的情况下进行培养。如果您的阳性对照相对于阴性对照没有明显变化，尝试着增加染料浓度和标记时间。我们的实验指南给出了启动建议。进行活细胞成像时尽可能得快。只有两种染料（CellROX Green和CellROX Deep Red）经甲醛固定处理后能保留在样品中。最后，确保您使用的滤光片和仪器设置与染料的激发和发射光谱相匹配。
It has been done. The problem is that plate readers are less sensitive than microscopes, with far less signal-to-background difference. It is worth trying, but first optimize concentrations and loading times with control cells, use a plate with little to no autofluorescence, and possibly optimize the gain setting in order to get the best signal possible. But don't expect the same sensitivity, even with optimization.
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This is not recommended as the two dyes overlap in the emission wavelength. There are other ROS reagents available in different wavelengths, such as CellROX Deep Red, which emits in the far-red range (665 nm), or dihydroethidium, which is emits in the visible red range (620 nm).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
H2DCFDA and similar derivatives are not fixable. The same goes for dihydroethidium and dihydrorhodamine. However, CellROX Deep Red and CellROX Green are retained for a limited time upon fixation with formaldehyde. CellROX Green may be retained upon subsequent Triton X-100 permeabilization. Avoid the use of any acetone or alcohol-based fixatives or fixatives that include alcohol, such as formalin.
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Many dyes that are used on mammalian cells have also been shown to be useful in bacterial cells. For example, CellROX Deep Red Reagent has been shown to work in B. subtilis (see Reference: http://www-brs.ub.ruhr-uni-bochum.de/netahtml/HSS/Diss/RaatschenNadja/diss.pdf). If you are interested in a particular dye, but are not sure if it will work on your bacteria, literature searches are the best way to check to see if it has been tested. If not, then it may be worth testing yourself.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
-Calcium flux: Each of the Oregon Green calcium indicators binds intracellular calcium with increasing affinity, providing a sensitivity range to match many applications. Oregon Green probes emit green fluorescence at resting levels of Ca2+ and increase their fluorescence intensity 14-fold with increasing Ca2+ concentration. The cell-permeant formulation (Cat. No. O6807) can be loaded in cell media and is compatible with flow cytometry.
-Rhodamine-based calcium indicators comprise a range of probes for large or small changes in Ca2+ concentration. They exhibit a 50-fold increase in fluorescence upon calcium binding and offer a range of wavelengths that can be used in conjunction with GFP or green-fluorescent dyes for multiplexing. Rhod-2, AM (Cat. No. R1245MP), in particular, localizes to mitochondria and can be used with flow cytometry.
-Membrane potential: A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. We offer a range of products specifically designed to assay mitochondrial membrane potential in live cells by flow cytometry, with minimal disruption of cellular function. The MitoProbe family of mitochondrial stains (Cat. Nos. M34150, M34151, and M34152) provide quick, easy, and reliable flow cytometric detection of the loss of mitochondrial membrane potential that occurs during apoptosis. MitoTracker dyes (Cat. Nos. M7510 and M7512) are membrane potential-dependent probes for staining mitochondria in live cells. The staining pattern of MitoTracker dyes is retained throughout subsequent flow cytometry immunocytochemistry, DNA end labeling, in situ hybridization, or counterstaining steps. The Mitochondrial Permeability Transition Pore Assay (Cat. No. M34153) provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. The mitochondrial permeability transition pore (MPTP) is a non-specific channel formed by components from the inner and outer mitochondrial membranes, and appears to be involved in the release of mitochondrial components during cell death.
-Phagocytosis: In phagocytosis, cells internalize particulate matter such as microorganisms, and this process is important for immune responses and during the clearance of apoptotic cells. Probes for studying phagocytosis include BioParticles indicators—bacteria and yeast labeled with fluorescent dyes.
-Tracking phagocytosis using a quench/wash-based assay can report on simple uptake, or a pH indicator can be used to monitor stages in the pathway. We have no-wash assays labeled with pHrodo Red or Green (Cat. Nos. A10010, P35361, P35364, P35365, P35366, and P35367) and no-wash assays for whole blood (Cat. Nos. A10025, A10026, P35381, and P35382), all suitable for flow cytometry.
-pH changes: Sensitive pH determinations can be made in a physiological range using either fluorescent intensity or ratiometric measurements. pHrodo dyes (Cat. Nos. P35373 and P35372) provide signal intensity modulation from pH 2 to pH 9 and with a choice of fluorescent wavelengths. Tracking internalization of fluorescent dextran is a routine method for analyzing pH changes in cellular compartments. Dextran conjugates of pHrodo dyes (Cat. Nos. P35368 and P10361) provide the most complete solution by allowing discrimination of vesicles from early endosomes to lysosomes, with no quench or wash required.
-Reactive oxygen species: Cells that are environmentally stressed usually contain greatly increased levels of reactive oxygen species (ROS). CellROX reagents are fluorogenic probes developed for the detection and quantitation of ROS in live cells. These cell-permeant reagents are non-fluorescent or very weakly fluorescent in the reduced state; however, when oxidized, they become brightly fluorescent and remain localized within the cell. We offer CellROX Green (Cat. No. C10492), CellROX Orange (Cat. No. C10493), and CellROX Deep Red (Cat. No. C10491) Assay Kits validated for flow cytometry.
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First, make sure you have both a negative (untreated) and positive (ROS-induced) sample to compare. A good positive control can be the use of 100 µM menadione for one hour or 50 µM nefazodone for 24 hours. H2O2 can also be used, though it does not work well for CellROX dyes. Some dyes, such as H2DCFDA, require esterase cleavage, so don't incubate in the presence of serum (which contains esterases that can prematurely cleave the dye). If your positive control does not show significant change compared to the negative control, try increasing the concentration and label time for the dye. Our manuals give starting recommendations. Be sure to image your live cells as soon as possible. Only two dyes (CellROX Green and CellROX Deep Red) are retained with formaldehyde fixation. Finally, make sure you are using filters and instrument settings to match the excitation and emission spectra of the dye.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.