A simple non-destructive method for the fixation and immunostaining of cultured cells encapsulated in alginate.
In the present paper we report a simple non-destructive method for the analysis of cells and their proteins encapsulated in alginate. Investigations with a co-culture of the human hepatoma cell line HepG2 and primary human fibroblasts are reported. We studied the expression of three proteins, namely intracellular alpha-foetoprotein and extracellular albumin, expressed by HepG2, and membrane-bound CD90 (Thy-1), expressed by the fibroblasts. Fluorimetric and colorimetric staining methods were evaluated and compared. We optimized the method by investigating a range of concentrations of the washing buffers. The technique protected the capsule and cell structures and enabled the investigated proteins to be clearly and reliably visualized within the encapsulated co-culture in their respective intracellular, membrane-bound and extracellular domains. These results demonstrate that this simple method is suitable for the non-destructive analysis of protein expression by cells encapsulated in alginate.
Department of Cell Technology and Applied Stem Cell Biology Biotechnology and Biomedicine Centre, BBZ, University of Leipzig, Deutscher Platz 5, 04103, Leipzig, Federal Republic of Germany.
Aldo
A
Leal-Egana
J Michael
JM
Heinrich
Mark D
MD
Smith
Marcin
M
Nowicki
Augustinus
A
Bader
D000418:Albumins / Q000096:biosynthesis; D000464:Alginates / Q000737:chemistry*; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D005347:Fibroblasts / Q000737:chemistry / Q000648:ultrastructure*; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D018800:Thy-1 Antigens / Q000096:biosynthesis; D016707:Tissue Fixation / Q000379:methods*; D000509:alpha-Fetoproteins / Q000096:biosynthesis
D000418:Albumins; D000464:Alginates; D006603:Hexuronic Acids; D018800:Thy-1 Antigens; D000509:alpha-Fetoproteins; D020723:Glucuronic Acid