Wilson H A, Greenblatt D, Taylor C W, Putney J W, Tsien R Y, Finkelman F D, Chused T M
J Immunol. 1987 Mar 15;138(6):1712-8.
We report the cytosolic free calcium, [Ca2+]i, responses of single murine B lymphocytes to whole and F(ab')2 fragments of anti-Ig measured in the flow cytometer with indo-1, a new fluorescent chelator of calcium. The principle advantages of this recording system are these: Indo-1 is highly fluorescent; hence, loading concentrations that introduce artifacts in the reported [Ca2+]i signal may be avoided. The measurement of [Ca2+]i by fluorescence ratio corrects for nonuniform dye uptake, making possible quantitative estimates of [Ca2+]i in single cells and an assessment of the variability of population responses. Baseline recordings of unstimulated lymphocytes indicated a narrow, stable range of [Ca2+]i (75 to 125 nM). The [Ca2+]i rise induced by various anti-Ig preparations exhibited considerable heterogeneity. The initial mean value for F(ab')2 anti-Ig-stimulated cells peaked above 1 microM and was due only to the release of Ca2+ from intracellular stores. A steady state elevation of [Ca2+]i was reached by 5 min and persisted for hours. Cells stimulated with intact anti-Ig reached similar initial peak [Ca2+]i values, but then declined toward baseline. This difference was due to membrane Ig-IgG Fc receptor (mIg-Fc gamma R) cross-linkage, because blocking the Fc gamma R with a monoclonal antibody made the [Ca2+]i responses to F(ab')2 and intact anti-Ig identical. The attenuation of the [Ca2+]i signal by mIg-Fc gamma R cross-linkage is proceeded by a corresponding Fc gamma-mediated reduction in anti-Ig-induced inositol trisphosphate elevation. These findings outline a biochemical basis for mIg- and Fc gamma R-mediated activation and regulation intrinsic to the B cell, and demonstrate the advantages of indo-1 over quin2 for fluorescent measurement of [Ca2+]i in small cells.
我们报告了用新型钙荧光螯合剂indo-1在流式细胞仪中测量的单个鼠B淋巴细胞对完整抗Ig和抗Ig F(ab')2片段的胞质游离钙（[Ca2+]i）反应。该记录系统的主要优点如下：indo-1具有高荧光性；因此，可以避免引入会在报告的[Ca2+]i信号中产生伪影的加载浓度。通过荧光比率测量[Ca2+]i可校正染料摄取不均匀的问题，从而能够对单个细胞中的[Ca2+]i进行定量估计，并评估群体反应的变异性。未刺激淋巴细胞的基线记录表明[Ca2+]i处于狭窄、稳定的范围（75至125 nM）。各种抗Ig制剂诱导的[Ca2+]i升高表现出相当大的异质性。F(ab')2抗Ig刺激的细胞的初始平均值峰值高于1 microM，且仅由于细胞内储存的Ca2+释放所致。5分钟时达到[Ca2+]i的稳态升高，并持续数小时。用完整抗Ig刺激的细胞达到相似的初始峰值[Ca2+]i值，但随后降至基线。这种差异是由于膜Ig-IgG Fc受体（mIg-FcγR）交联所致，因为用单克隆抗体阻断FcγR会使对F(ab')2和完整抗Ig的[Ca2+]i反应相同。mIg-FcγR交联导致的[Ca2+]i信号衰减伴随着Fcγ介导的抗Ig诱导的肌醇三磷酸升高相应降低。这些发现概述了B细胞固有的mIg和FcγR介导的激活和调节的生化基础，并证明了indo-1在小细胞中荧光测量[Ca2+]i方面优于quin2。
