Novel application of a fibrin cell block method to evaluate melanocytic cell populations.
Confirming melanocytic lineage and purity is important for experiments using cultured human melanocytes. The objective of this study was to develop a simple, reliable method to evaluate and archive cultured melanocytic cells. Melanocytes were isolated from adult skin biopsies or from neonatal foreskins using standard culturing methods. Fibrin cell blocks (FCBs) were prepared from cultured cells at passages two and six. Fibrin blocks were paraffin-embedded and sectioned for immunohistochemical (CD68, Melan-A, and HMB-45) and H & E staining. Flow cytometry was performed (Melan-A) at passage six. A mixing experiment with cultured melanocytes and fibroblasts was performed and cell population purity was determined by manual counts of positively staining cells in the FCBs and by flow cytometry. The FCB method of evaluating population purity was validated experimentally and by correlation with flow cytometry results. Preparation of a FCB followed by immunohistochemical staining is an easy and inexpensive way to confirm melanocytic lineage, estimate population purity, and provide a permanent archive of cultured cells.
Florell
Scott R
SR
Department of Dermatology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA. scott@derm.med.utah.edu
Schmidt
Soo J
SJ
Porter-Gill
Patricia
P
Albertine
Kurt H
KH
Murphy
Kelley J
KJ
McKinney
Courtney B
CB
Boucher
Kenneth M
KM
Grossman
Douglas
D
Biddle
Diana L
DL
Clayton
Frederic
F
Layfield
Lester J
LJ
Leachman
Sancy A
SA
D000328:Adult; D015703:Antigens, CD / Q000276:immunology; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology; D000951:Antigens, Neoplasm; D002478:Cells, Cultured; D005337:Fibrin / Q000737:chemistry*; D005347:Fibroblasts / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D058965:MART-1 Antigen; D008544:Melanocytes / Q000166:cytology*; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins / Q000276:immunology; D012867:Skin / Q000166:cytology*; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000951:Antigens, Neoplasm; D005337:Fibrin; D058965:MART-1 Antigen; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C548667:MLANA protein, human