Routine flow cytometric immuno-staining of T-cell perforin is preserved using diethylene glycol for erythrocyte-lysis but lost by the use of ammonium chloride.
The system of perforin-containing lytic granules of cytotoxic lymphocytes plays an important role in the immune defense machinery. Investigating the capacity and efficacy of this system in and ex vivo is helpful to understand immune responses and their modulation by therapeutic interventions. With regard to its pathophysiological function, we recently demonstrated a substantial increase of perforin-positive CD8+ T cells in the peripheral blood of patients with acute exacerbated psoriasis and severe generalized drug reactions, and, in marked contrast, a highly significant perforin-depletion and a perforin-hyperreleasability in atopic dermatitis (AD). To streamline the perforin staining procedure, isolation of peripheral blood mononuclear cells (PBMC) by Ficoll density centrifugation was to be replaced by lysis of erythrocytes. Ammonium chloride lysis, however, reduced the perforin content of CD8+ T cells substantially (up to 75-100%) as compared with Ficoll isolation of PMC. Incubation of cells in concanamycin A, a selective inhibitor of H+-ATPases, resulted in a similar loss of perforin staining pointing to the critical influence of lysosomal pH. Using diethylene glycol-mediated erythrocyte lysis, perforin was well preserved to be readily detectable by immuno flow cytometry. Representative examples of the application of this optimized perforin staining procedure as well as accumulated data are given for various dermatological disorders (psoriasis, atopic dermatitis, cutaneous drug reactions, graft-versus-host disease (GVHD) with strong involvement of the cytotoxic T-cell population. Our findings may help to explain recent conflicting reports about a widely varying range of the portion of perforin-positive cells in healthy individuals as a reflection of such artificial methodological influences.
Ambach
Andreas
A
Department of Dermatology and Venereology, Otto-von-Guericke-University, Magdeburg, Germany. Andreas.Ambach@Medizin.Uni-Magdeburg.de
Bonnekoh
Bernd
B
Gollnick
Harald
H
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000643:Ammonium Chloride / Q000494:pharmacology*; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D002469:Cell Separation; D004912:Erythrocytes / Q000378:metabolism*; D005026:Ethylene Glycols / Q000737:chemistry*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000737:chemistry*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D013601:T-Lymphocytes / Q000378:metabolism*
D005026:Ethylene Glycols; D018942:Macrolides; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D000643:Ammonium Chloride; D054353:Perforin; C013484:diethylene glycol; C036978:concanamycin A; D000251:Adenosine Triphosphatases