Rhod-3 钙成像试剂盒

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。

This is a known drawback of Calcium Crimson, AM (as well as Calcium Orange, AM and Fura Red, AM, which are also in the same emission range). You can try increasing the concentration and washing out of any unlabeled dye from the media, to try to get better signal-to-background. If that fails, we recommend using Rhod-3 AM instead, which has a much better change in signal in that wavelength.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.