Expansion of cultured U2Os cell was performed according to the protocol from M'Saad and Bewersdorf (2020) (link). In short, unstained U2Os cells were fixed with 4% paraformaldehyde for 15 min., washed with PBS and post-fixed with a 0.7% paraformaldehyde/1% Acrylamide solution. The fixed cells were embedded in a thin layer of gel (19% Sodium Acrylate, Chem Cruz, catalogue number sc236893; 10% Acrylamide, Sigma-Aldrich, catalogue number 1.19784.0100; 0.1% N,N′-Cystamine-bisAcrylamide, Sigma-Aldrich catalogue number 294381; 0.25% Ammonium persulfate, Fisher BioReagents, catalogue number 87687; and 0.25% N,N,N,N′-tetramethylethylenediamine, Sigma-Aldrich, catalogue number A4929).
The gel was allowed to polymerize for 1 h at 37°C. The cells in the gel were denatured in 1 ml denaturation buffer (200 mM SDS, 200 mM Sodium Chloride, 50 mM Tris-HCl pH 6.8) at 73°C for 1 h. After denaturation, all proteins in the gel were stained with 20 μg/ml NHS-ATTO594 (Sigma-Aldrich, catalogue number 08741) in 100 mM bicarbonate for 1.5 h. The gel was expanded in milli-Q water overnight. Afterwards the Gel was expanded in milli-Q water overnight. The resulting cells were expanded approximately seven times.
The gel was allowed to polymerize for 1 h at 37°C. The cells in the gel were denatured in 1 ml denaturation buffer (200 mM SDS, 200 mM Sodium Chloride, 50 mM Tris-HCl pH 6.8) at 73°C for 1 h. After denaturation, all proteins in the gel were stained with 20 μg/ml NHS-ATTO594 (Sigma-Aldrich, catalogue number 08741) in 100 mM bicarbonate for 1.5 h. The gel was expanded in milli-Q water overnight. Afterwards the Gel was expanded in milli-Q water overnight. The resulting cells were expanded approximately seven times.
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