为什么我看不到活细胞荧光指示剂信号有明显变化?
不管何种活细胞指示剂染料(如钙指示剂,pH指示剂,金属离子指示剂),请务必确保上样过程中无血清,否则血清会过早地切割带有AM酯基的染料并非特异性结合染料。在检测样品之前,请使用阳性对照优化染料浓度和染色时间来得到最佳的信号与背景比值。做阳性对照时缓冲液一定要包含已知浓度的自由离子和用于打开离子通道的离子载体(例如Fluo-4 AM一类的钙指示剂,它包括缓冲液中加入钙与卡西霉素,又比如pH指示剂,不同pH值的缓冲液与尼日利亚霉素相结合)。类似于CellROX Green 或H2DCFDA需要一个细胞活性氧(ROS)刺激作为阳性对照,例如甲萘醌。最后,确保成像系统具有灵敏的探测器。例如相对于显微镜或流式细胞仪,读板仪检测信号和背景差异的能力就比较低。
Can I fix my cells after loading with Fluo-4 AM dye for the detection of calcium?
No. Since Fluo-4 AM isn't covalently bound to any cellular components and fixation compromises the membrane, the dye would not be retained by the cell.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
I am doing calcium flux imaging with your Fura-2 calibration kit, but am seeing a large variability in ratio in different places around the slide. I am correcting for uniform illumination, using the product as directed, and sealing the coverslip with nail polish.
The nail polish may be the problem. The Kd value (calcium sensitivity) changes depending upon the dye's environment. Nail polish has solvents that can leech under the coverslip and cause variability. We recommend either going without a sealing or sealing with melted paraffin painted on the coverslip edges with a cotton-tipped applicator (paraffin is hydrophobic and has no solvents).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
I need to label cells with Fluo-4, AM, for a calcium flux assay. How long after labeling will the dye be retained?
After loading dye into the cells, intracellular esterases remove the 'AM' moiety from the dye. When the 'AM' group is removed, the dye is able to bind calcium and fluoresce. Since the dye is not covalently bound to any cellular components, it may be actively effluxed from the cell. The rate of efflux is dependent upon the inherent properties of the cell, culture conditions and other factors. The dye may be retained for hours, days or even weeks or lost in a matter of minutes. The use of Probenecid (Cat. No. P36400) limits loss by active efflux.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Why don't I see a significant change in signal for my live-cell fluorescent indicator dye?
Regardless of the type of live-cell indicator dye (e.g., calcium indicators, pH indicator, metal ion indicators), make sure there is no serum during the loading step, which can prematurely cleave dyes with AM esters and bind dyes non-specifically. Always optimize the dye concentration and staining time with a positive control before you run your test samples, to give the best signal-to-background. Always run a positive control with a buffer containing free ions of known concentration and an ionophore to open pores to those ions (for instance, for calcium indicators like Fluo-4 AM, this would include a buffer with added calcium combined with calcimycin, or for pH indicators, buffers of different pHs combined with nigericin). Reactive oxygen indicators, such as CellROX Green or H2DCFDA would require a cellular reactive oxygen species (ROS) stimulant as a positive control, such as menadione. Finally, make sure your imaging system has a sensitive detector. Plate readers, for instance, have much lower detector efficiency over background, compared to microscopy or flow cytometry.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.