The dox-inducible A673/TR/shEF cell line was previously described.40 (link) Knockdown of EWS-FLI1 was achieved by addition of 1 μg/ml dox to the medium (24–72 h). The SK-N-MC EwS cell line was kindly provided by J Biedler (Memorial Sloan-Kettering Cancer Centre, New York, NY, USA). Cell lines were authenticated by STR profiling and regularly tested for mycoplasma (Mykoalert detection kit; Lonza, Basel, Switzerland). For serum induction cells were starved overnight in 0.2% fetal bovine serum medium with subsequent serum stimulation for 60 min with 20% fetal bovine serum medium. Latrunculin B (Cayman Chemicals, Ann Arbor, MI, USA) was added in serum-free DMEM at 1 μM concentration to the overnight starved cells 30 min before serum induction. Vehicle controls were treated with dimethyl sulfoxide (<0.05%).
Mykoalert detection kit
Manufactured by Lonza
Sourced in Switzerland
The Mykoalert detection kit is a laboratory equipment designed for the detection of specific analytes. It provides a reliable and efficient method for the identification and quantification of the target analytes. The kit includes all the necessary components and reagents required for the analysis process.
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2 protocols using mykoalert detection kit
1
Inducible EWS-FLI1 Knockdown in A673/TR/shEF Cells
2
Inducible EWS-FLI1 Knockdown in Ewing Sarcoma Cells
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A673/TR/shEF and shSKE-17T cell clones harbouring dox-inducible sh-EWS-FLI1 constructs were derived from A673 and SK-N-MC parental cell lines, and were kindly provided by J. Alonso (Instituto de Investigación de Enfermedades Raras, Madrid, Spain) and O. Delattre (Institute Curie, Paris, France)5 (link),59 (link). SKshFli1#3 cell clone carrying a dox-inducible shRNA construct targeting the 3’UTR of FLI1 (5’-TTATTCATCTCTTTGTTCAGGT-3’; pRSIT-U6Tet-shRNA-PGK-TetRep-2A-GFP-2A-puro vector plasmid; Cellecta Inc., Mountain View, CA, USA) was generated from SK-N-MC parental cell line. TC32/223 cells were a kind gift of David McFadden from UT Southwestern, Houston, Texas. Knockdown of EWS-FLI1 by shRNA induction was achieved by addition of 1 μg/ml dox (A673/TR/shEF, shSKE-17T, TC32/223) or 100 ng/ml dox (SKshFli1#3) to the medium. Cell lines were authenticated by STR profiling and were regularly screened for mycoplasma (Mykoalert detection kit; Lonza, Basel, Switzerland).
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