Electrical & Computer Engineering, University of Virginia, Charlottesville, VA, 22904, USA.
Chemistry, University of Virginia, Charlottesville, VA, 22904, USA.
Biosens Bioelectron. 2022 Aug 15;210:114346. doi: 10.1016/j.bios.2022.114346. Epub 2022 May 7.
Measurement of macrophage activation and its modulation for immune regulation is of great interest to arrest inflammatory responses associated with degeneration of intervertebral discs that cause chronic back pain, and with transplants that face immune rejection. Due to the phenotypic plasticity of macrophages that serve multiple immune functions, the net disease outcome is determined by a balance of subpopulations with competing functions, highlighting the need for single-cell methods to quantify heterogeneity in their activation phenotypes. However, since macrophage activation can follow several signaling pathways, cytometry after fluorescent staining of markers with antibodies does not often provide dose-dependent information on activation dynamics. We present high throughput single-cell impedance cytometry for multiparametric measurement of biophysical changes to individual macrophages for quantifying activation in a dose and duration dependent manner, without relying on a particular signaling pathway. Impedance phase metrics measured at two frequencies and the electrical diameter from impedance magnitude at lower frequencies are used in tandem to benchmark macrophage activation by degenerated discs against that from lipopolysaccharide stimulation at varying dose and duration levels, so that reversal of the activation state by curcumin can be ascertained. This label-free single-cell measurement method can form the basis for platforms to screen therapies for inflammation, thereby addressing the chronic problem of back pain.
测量巨噬细胞的激活及其调节对于抑制与椎间盘退变相关的炎症反应、抑制免疫排斥反应非常重要，而椎间盘退变会引起慢性背痛，免疫排斥反应则会导致移植失败。由于巨噬细胞具有多种免疫功能的表型可塑性，因此净疾病结局取决于具有竞争功能的亚群之间的平衡，这突出表明需要单细胞方法来量化其激活表型的异质性。然而，由于巨噬细胞的激活可以遵循几种信号通路，因此用抗体对荧光标记物进行细胞术染色后，通常无法提供关于激活动力学的剂量依赖性信息。我们提出了高通量单细胞阻抗细胞术，用于对单个巨噬细胞的生物物理变化进行多参数测量，以便以剂量和时间依赖性方式定量测量激活，而无需依赖特定的信号通路。在两个频率下测量的阻抗相位指标以及在较低频率下的阻抗幅度的电直径被串联使用，以将退变椎间盘引起的巨噬细胞激活与不同剂量和时间水平的脂多糖刺激引起的激活进行基准比较，从而确定姜黄素对激活状态的逆转作用。这种无标记的单细胞测量方法可以为筛选炎症治疗方法的平台提供基础，从而解决慢性背痛这一难题。
