Department of Science and Environment, Enhanced Perioperative Oncology (EPeOnc) Consortium, Roskilde University, Universitetsvej 1, 4000, Roskilde, Denmark; Center for Surgical Science, Enhanced Perioperative Oncology (EPeOnc) Consortium, Department of Surgery, Zealand University Hospital, Lykkebækvej 1, 4600, Køge, Denmark.
Department of Science and Environment, Enhanced Perioperative Oncology (EPeOnc) Consortium, Roskilde University, Universitetsvej 1, 4000, Roskilde, Denmark.
Anal Biochem. 2022 Aug 1;650:114723. doi: 10.1016/j.ab.2022.114723. Epub 2022 May 12.
Methods measuring cell proliferation and adhesion are widely used but each hold limitations. We, therefore, introduce novel methods for measuring cell proliferation and adhesion based on CRISPR-modified cancer cell lines secreting luciferase to the growth media.
Using CRISPR genome editing, we generated stable luciferase-secreting LS174T, HCT 116, Caco-2, and PANC-1 cell lines. The modified cells were seeded, and luciferase activity was measured in the media and compared to Coulter counter cell counts and iCELLigence impedance assay to evaluate the value of the secreted luciferase activities as a measurement for adhesion and proliferation.
Our results demonstrate that luciferase secreted into the media can be used quantifying cell proliferation and adhesion. The adhesion luciferase assay and the iCELLigence impedance assay showed similar results with increased significant difference observed in the luciferase assays. The luciferase proliferation assay showed increased growth following increased serum concentrations in all cell lines vs. only two cell lines in the iCELLigence impedance assay.
Our results show that the luciferase adhesion and proliferation assays are reliable methods for measuring adhesion and proliferation. The luciferase assays have advantages over existing assays as they are highly sensitive, easy to perform, non-invasive and suitable as high-throughput measurements.
细胞增殖和黏附的测量方法被广泛应用，但每种方法都有其局限性。因此，我们引入了基于 CRISPR 修饰的分泌荧光素到生长培养基中的癌细胞系的新型细胞增殖和黏附测量方法。
我们使用 CRISPR 基因组编辑，生成了稳定分泌荧光素的 LS174T、HCT116、Caco-2 和 PANC-1 细胞系。将修饰后的细胞播种，测量培养基中的荧光素活性，并与库尔特计数器细胞计数和 iCELLigence 阻抗测定法进行比较，以评估分泌的荧光素活性作为黏附和增殖测量的价值。
我们的结果表明，分泌到培养基中的荧光素可用于定量细胞增殖和黏附。黏附荧光素测定法和 iCELLigence 阻抗测定法的结果相似，在荧光素测定法中观察到显著增加的差异。与 iCELLigence 阻抗测定法中仅两种细胞系相比，在所有细胞系中，随着血清浓度的增加，荧光素增殖测定法显示出更高的生长。
我们的结果表明，荧光素黏附测定法和增殖测定法是测量黏附和增殖的可靠方法。与现有测定法相比，荧光素测定法具有高度灵敏、易于操作、非侵入性和适合高通量测量的优点。
