Effective enrichment of murine epidermal Langerhans cells by a modified(mismatched) panning technique.
A method for the enrichment of murine epidermal Langerhans cells (LC) is described in detail. It is based on positive selection of LC from pre-enriched fresh or cultured epidermal cell suspensions derived from ear skin by a modified panning technique. The method uses the interspecies cross-reactivities of anti-immunoglobulin antibodies: when LC in an epidermal cell suspension are labeled with mouse anti-major histocompatibility complex (MHC) class II antibodies they bind to petri dishes coated with anti-rat immunoglobulin antibodies. We therefore call this method "mismatched panning." After rinsing off non-adherent cells, the adherent LC can easily be dislodged by adding excess amounts of rat immunoglobulins, which effectively compete with the LC-bound mouse anti-MHC class II antibodies for binding to the petri dish. Using this modified panning technique, both fresh and cultured LC could be enriched up to more than 90% purity. From one ear, 2.0-3.0 x 10(4) fresh LC and 3.0-4.5 x 10(4) cultured LC could be obtained. Of all LC present in a primary, unenriched epidermal cell suspension, 40-60% were recovered when panned immediately after isolation of the epidermal cells and 50-75% when panned after 3 d of epidermal cell culture. Viability of panned LC was consistently more than 90%. Antigen presenting and T-cell-stimulating capacity of LC and responses to the cytokines granulocyte/macrophage colony-stimulating factor and tumor necrosis factor-alpha were not impaired by this panning procedure. The major advantage of this method compared to pre-existing panning techniques is the ease with which adherent LC can be dislodged from the panning dishes. Because the elution procedure is very gentle, virtually all panned LC are viable. As a consequence, good yields of highly enriched LC can be obtained in a reasonable time.
Koch
F
F
Department of Dermatology, University of Innsbruck, Austria.
Kämpgen
E
E
Schuler
G
G
Romani
N
N
D000818:Animals; D000938:Antigen-Presenting Cells / Q000166:cytology / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D000078404:Epidermal Cells; D000949:Histocompatibility Antigens Class II / Q000032:analysis; D007801:Langerhans Cells / Q000166:cytology* / Q000502:physiology; D008285:Major Histocompatibility Complex / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H
D016207:Cytokines; D000949:Histocompatibility Antigens Class II