Department of Rheumatology and Clinical Immunology, Charité University Hospital, Berlin, Germany.
J Fluoresc. 2013 May;23(3):509-18. doi: 10.1007/s10895-013-1163-4. Epub 2013 Feb 14.
Flow cytometry has become a widely-used and powerful tool for the characterization of cells according to their expression of specific proteins. However, sensitivity of this method is still limited since conventionally labeled antibodies can be conjugated with at maximum 1-10 dye molecules. This fact resulted in the need to develop new techniques in order to identify molecules which are expressed in very low but functionally relevant amounts. In the past, we have successfully used a liposome-based high-sensitivity immunofluorescence technique to measure the expression of low abundant membrane bound glucocorticoid receptors (mGR) on different cell types. The use of this technique allows the detection of as few as 50-100 antigen molecules per cell which is due to a 100-fold to 1000-fold increase in fluorescence signal intensity compared with conventional methods. The higher sensitivity is achieved since thousands of dye molecules can be enclosed in liposomes. Another modern high-sensitivity immunofluorescence staining method is the purchasable Fluorescence Amplification by Sequential Employment of Reagents (FASER) procedure. Here, we aimed at comparing sensitivity and specificity of these two techniques for the detection of the mGR. Our data demonstrate the FASER technique to be more sensitive and also more specific for the detection of mGR as compared to the liposome technique. However, both methods have advantages and disadvantages which are discussed in detail.
流式细胞术已成为一种广泛使用且功能强大的工具，可根据细胞表达特定蛋白质的情况对其进行特征描述。然而，由于传统标记的抗体最多只能与 1-10 个染料分子结合，因此该方法的灵敏度仍然有限。这一事实促使人们需要开发新技术，以鉴定在非常低但功能相关的数量下表达的分子。过去，我们成功地使用基于脂质体的高灵敏度免疫荧光技术来测量不同细胞类型中低丰度膜结合糖皮质激素受体 (mGR) 的表达。由于与传统方法相比，荧光信号强度增加了 100 倍至 1000 倍，因此该技术可以检测到每个细胞中低至 50-100 个抗原分子。由于可以将数千个染料分子包含在脂质体中，因此可以实现更高的灵敏度。另一种现代高灵敏度免疫荧光染色方法是可购买的荧光试剂顺序使用扩增（FASER）程序。在这里，我们旨在比较这两种技术检测 mGR 的灵敏度和特异性。我们的数据表明，与脂质体技术相比，FASER 技术在检测 mGR 方面更灵敏，也更具特异性。然而，这两种方法都有各自的优缺点，我们对此进行了详细讨论。
