Evaluating TNBC Tumorsphere Viability

The TNBC cells were cultured in NanoCulture plates (2 × 10⁴/well, SCIVAX Corporation, Kanagawa, Japan) and 96-well ultralow attachment plates (4 × 10³/well, Costar^®, New York, NY, USA). Cells transfected with 10 nM scramble or siRNA against DYRK1B were cultured for 7 d to observe tumorsphere of the control and silenced tumorsphere under microscope. Tumorsphere viability was evaluated by 3D CellTiter Glo (G9681, Promega) or Calcein AM (Green)/Ethidium homodimer-1 (EthD-1, Red) (LIVE/DEAD^® Viability/Cytotoxicity Kit, ThermoFisher Scientific) to differentiate live and dead cells. Tumorsphere viability was evaluated and quantitated by Fluoroskan Ascent FL reader (Thermo Fisher Scientific), as previously described [17 (link)].

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Chang C.C., Chiu C.C., Liu P.F., Wu C.H., Tseng Y.C., Lee C.H, & Shu C.W. (2021). Kinome-Wide siRNA Screening Identifies DYRK1B as a Potential Therapeutic Target for Triple-Negative Breast Cancer Cells. Cancers, 13(22), 5779.

Publication 2021

96-well ultralow attachment plates 3D CellTiter Glo LIVE/DEAD® Viability/Cytotoxicity Kit Fluoroskan Ascent FL reader

Calcein Cells Cytotoxicity Ethd 1 Microscope Promega Sirna

Corresponding Organization : National Sun Yat-sen University

Other organizations : Kaohsiung Armed Forces General Hospital, Kaohsiung Medical University, Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung Veterans General Hospital, National Yang Ming University Hospital, National Yang Ming Chiao Tung University

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Variable analysis

independent variables

Transfection with 10 nM scramble or siRNA against DYRK1B

dependent variables

Tumorsphere formation
Tumorsphere viability (evaluated by 3D CellTiter Glo or Calcein AM/Ethidium homodimer-1)

control variables

TNBC cells cultured in NanoCulture plates (2 × 10^4/well) and 96-well ultralow attachment plates (4 × 10^3/well)
Cells cultured for 7 days

controls

Scramble siRNA as a negative control

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