Fernandez Josiah, Torchia Enrique C
Department of Dermatology and Charles C. Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus.
Department of Dermatology and Charles C. Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus;
J Vis Exp. 2019 May 9(147). doi: 10.3791/59353.
The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of >40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.
本实验方案的目的是利用从小鼠皮肤表皮分离的单细胞，以细胞周期依赖性方式检测和定量蛋白质表达变化。有七个重要步骤：从真皮中分离表皮、消化表皮、用顺铂对表皮细胞群体进行染色、样本条形码标记、用金属标记抗体对细胞周期标记物和感兴趣的蛋白质进行染色、通过质谱流式细胞术检测金属标记抗体以及分析不同细胞周期阶段的表达情况。与组织学方法相比，这种方法的优势在于有可能在单个细胞的不同细胞周期阶段检测>40种不同标记物的表达模式。这种方法还允许对蛋白质表达进行多变量相关分析，比组织学/成像方法更具可量化性。该实验方案的缺点是需要单细胞悬液，这导致组织切片染色提供的位置信息丢失。这种方法可能还需要加入额外的标记物以识别粗细胞悬液中的不同细胞类型。该实验方案在增生性皮肤病模型分析中具有明显的应用价值。此外，通过添加谱系特异性抗体，该实验方案可适用于特定细胞亚型（如干细胞）的分析。该实验方案也可适用于其他实验物种皮肤细胞的分析。
