A method for flow cytometric cell cycle analysis of normal and psoriatic human epidermis based on a detergent/citric acid technique for suspension of nuclei.
A new method is described for flow cytometric cell cycle analysis of normal and psoriatic human epidermis, based on non-enzymatic tissue disaggregation. The epidermis was isolated by treatment with acetic acid and stored by freezing. After thawing, the epidermis was disintegrated into a nuclear suspension by 3 steps: incubation with dithiotreitol, whirling in a buffer (pH 7.4) with the non-ionic detergent Nonidet P40, EGTA, RNase and spermine, and whirling after addition of citric acid to a final concentration of 1% (pH 2.4). The suspension was stained with propidium iodide and filtered before flow cytometry. The yield of suspended nuclei was approximately 70% of the original number of cells in the tissue. The detergent/citric acid method was found to be preferable to an ultrasonication method previously used on human epidermis. All cell cycle and cell maturation stages were represented in the detergent/citric acid suspension, in contrast to the selection of immature G1, S and G2 stages with enzymatic methods. In the analysis of psoriatic epidermis inadequately matured (parakeratotic) cells were present in the suspension and had to be discriminated by gating on light scattering intensity, as they were not susceptible to lysis and did not stain properly. The fraction of S phase nuclei was on average 1.9% in normal and 7.7% in psoriatic epidermis, thus confirming the results of other investigators using enzymes. The presence of mitotic figures in the suspension was demonstrated by flow sorting. In this way the mitotic fraction was estimated to 0.06% in normal and 0.22% in psoriatic epidermis, confirming histological data of other investigators.
Larsen
J K
JK
Frentz
G
G
Møller
U
U
Christensen
I J
IJ
D001706:Biopsy; D002453:Cell Cycle; D002467:Cell Nucleus / Q000473:pathology; D002951:Citrates; D003902:Detergents; D000078404:Epidermal Cells; D004817:Epidermis / Q000473:pathology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D011565:Psoriasis / Q000473:pathology*
D002951:Citrates; D003902:Detergents