期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 147, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/59353
关键词
Developmental Biology; Issue 147; Cell Cycle; Epidermis; Skin; Keratinocyte isolation; Mouse models; Mass cytometry; Proliferation; Correlation of protein expression; Single cell analysis
资金
- University of Colorado (UC) Skin Disease Center Morphology and Phenotyping Cores [NIAMS P30 AR057212]
- UC Cancer Center Flow Cytometry Shared Resource
- Department of Dermatology, the Gates Center for Regenerative Medicine at the University of Colorado
- [NCI P30 CA046934]
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.
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