细胞的固定和透化对于流式细胞术中标记细胞内生物标志物至关重要。然而，这些化学处理通常会改变脆弱的靶标，例如细胞表面和荧光蛋白，并可能破坏化学敏感的荧光标记。这会降低测量精度并影响样本工作流程，从而导致数据质量下降。在这里，我们展示了一种新颖的多通道流式细胞术方法来解决这个长期存在的问题。我们的技术利用带有激光粒子的单个细胞条形码，能够在保持单细胞分辨率的情况下对相同细胞进行连续分析。在破坏性样品处理之前测量化学脆性蛋白质标记物及其荧光染料缀合物，并在固定和透化后随后测量细胞内标记物。我们证明了我们的技术在准确测量细胞内荧光蛋白和甲醇敏感抗原和荧光团以及各种表面和细胞内标记方面的有效性。这种方法显着增强了分析的灵活性，能够实现准确、全面的细胞分析，而不受传统一次性测量流式细胞术的限制。这项创新为流式细胞术在免疫肿瘤学、干细胞研究和细胞生物学领域的广泛应用开辟了新途径。

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins, and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular fluorescent proteins and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cell analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.