Division of Education for Global Standard, Institute of Liberal Arts and Science, Kanazawa University, Kanazawa, Japan.
Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan.
Curr Protoc Cell Biol. 2020 Mar;86(1):e103. doi: 10.1002/cpcb.103.
We developed a modified invasion assay in three-dimensional (3D) gels that permits isolation of invading cells as living cells, termed an invading cell-trapping (iCT) assay. A small cell strainer consisting of nylon mesh with pores of 40-µm square is used in this assay. A layer of gel composed of extracellular-matrix components is formed on each side of the nylon mesh, which permits cell migration or invasion from one gel layer to the other. At the end of the assay, the two gel layers are removed from the apparatus and easily separated from each other. Invading cells from the primary gel are trapped in the secondary gel, which maintains the morphology and other properties of the invasive cells in a 3D matrix. The cells that have invaded are observed and counted with a standard light microscope without cell staining. There is no need for a specialized microscope, imaging analysis software, or advanced cell-biological technical knowledge in this assay. This assay can also reduce measurement of nonspecific cell invasion by monitoring the upward invasion of cells. The viability of both invading and non-invading cells trapped in the gels can be assessed by typical colorimetric assays, if desired. This assessment characterizes the total number of cells (invading and non-invading cells) and the ratio of invading cells to total cells. By repeating the iCT assay, further enrichment of invasive and noninvasive cells can be attained. Thus, this assay improves comparative analyses between invasive and noninvasive cells. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Measuring upward cell invasion into collagen gel Basic Protocol 2: Measuring cell invasion from Matrigel into collagen gel Basic Protocol 3: Isolation and enrichment of highly invasive cells.
我们开发了一种改良的三维（3D）凝胶侵袭测定法，可以分离出作为活细胞的侵袭细胞，称为侵袭细胞捕获（iCT）测定法。该测定法使用由 40-µm 见方的尼龙网组成的小细胞滤器。尼龙网的两侧形成一层由细胞外基质成分组成的凝胶层，允许细胞从一层凝胶迁移或侵袭到另一层凝胶。测定结束时，将两层凝胶从仪器上取下，很容易彼此分离。来自原发凝胶的侵袭细胞被困在继发凝胶中，在 3D 基质中保持侵袭细胞的形态和其他特性。用标准的光学显微镜观察和计数未染色的细胞，无需特殊显微镜、成像分析软件或先进的细胞生物学技术知识。该测定法还可以通过监测细胞向上侵袭来减少对非特异性细胞侵袭的测量。如果需要，还可以通过典型的比色法评估被困在凝胶中的侵袭和非侵袭细胞的活力。这种评估可以确定总细胞数（侵袭和非侵袭细胞）以及侵袭细胞与总细胞的比例。通过重复 iCT 测定法，可以进一步富集侵袭性和非侵袭性细胞。因此，该测定法可以提高侵袭性和非侵袭性细胞之间的比较分析。 © 2020 年 John Wiley & Sons, Inc. 基本方案 1：测量胶原凝胶中细胞向上侵袭 基本方案 2：测量 Matrigel 中细胞向胶原凝胶中的侵袭 基本方案 3：高度侵袭性细胞的分离和富集。
