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.
Leal-Egana
Aldo
A
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.
Heinrich
J Michael
JM
Smith
Mark D
MD
Nowicki
Marcin
M
Bader
Augustinus
A
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