The invention relates to the field of pharmaceuticals and biology and represents a method for obtaining a viable heterogeneous population of skin cells, which includes taking a skin biopsy at a depth of ...
FIELD: medicine.
SUBSTANCE: method for determining the subpopulation composition of skin cells and obtaining the skin cytoimmunogram comprises sampling a skin biopsy specimen to a depth of 2 mm, homogenizing the tissue in a 0.9% aqueous solution of sodium chloride at a temperature of +23…+25°C, extracting the homogenate, filtering the homogenate through an inert filtering partition with a pore diametre of 20 mcm, centrifuging the homogenate at 400 g for 5 minutes at a temperature +23…+25°C. After centrifugation, the viability of the skin cells is determined, the skin cells are incubated for 20 minutes with monoclonal antibodies fluorochrome-labeled for flow cytometry, phenotyping of the skin cells is carried out, the number of skin cells of a specific phenotype - skin cytoimmunogram - are determined: keratinocytes CD49f+, among them activated CD49f+HLA-DR+; fibroblasts (fibrocytes) CD45-CD14-CD44+, among them activated CD45-CD14-CD44+CD80+; mast cells CD249+, among them activated CD249+CD63+; monocytes (macrophages) CD45+CD14+, among them activated CD45+CD14+HLA-DR+; intra-epidermal macrophages CD207+, among them activated CD207+CD80+, CD207+HLA-DR+, CD207+CD80+HLA-DR+; endothelial cells CD146+, among them activated CD146+CD34+, CD146+HLA-DR+, CD146+CD54+, CD146+CD54+HLA-DR+; lymphocyte populations: T-lymphocytes CD45+CD3+, T-helpers CD45+CD3+CD4+CD8-, T-suppressors CD45+CD3+CD4-CD8+, B-lymphocytes CD45+CD3-CD19+, NK-lymphocytes CD45+CD3-CD16+CD56+.
EFFECT: application of this method allows to make a quantitative and functional characteristic of all the skin constituent elements for use in diagnosis.
