Soft Agar Colony Formation Assay

The soft agar assay was performed as previously described (19 (link)). The base layer of each well consisted of 1.5 mL of serum-free 2X DMEM (Welgene, LM 201-50) with a final concentration of 0.5% Noble agar (BD Biosciences). After bottom agar solidification, 1.5 mL of 0.35% agar containing NIH3T3 cells (20,000) was seeded on the bottom agar layer and incubated for 14 days. Medium was changed every 2 days for 2 weeks. Colonies were fixed with 4% paraformaldehyde and then stained with 0.05% crystal violet (Sigma-Aldrich, C6158).

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An S., Koh H.H., Chang E.S., Choi J., Song J.Y., Lee M.S, & Choi Y.L. (2022). Unearthing novel fusions as therapeutic targets in solid tumors using targeted RNA sequencing. Frontiers in Oncology, 12, 892918.

Publication 2022

LM 201-50 Noble agar C6158

Agar Assay Crystal violet Nih3t3 cells Paraformaldehyde Serum

Corresponding Organization : Sungkyunkwan University

Other organizations : Yonsei University, Severance Hospital

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Variable analysis

independent variables

Cells used (NIH3T3 cells)

dependent variables

Colony formation

control variables

Serum-free 2X DMEM (Welgene, LM 201-50)
0.5% Noble agar (BD Biosciences)
0.35% agar
Incubation period (14 days)
Medium change frequency (every 2 days for 2 weeks)

controls

Positive control: Not specified
Negative control: Not specified

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