Propagating PDX Neurospheres for Drug Screening

PDXs were propagated as neurospheres, cultured in PDX media (DMEM/F12) (50/50 with 2% B27, 20 ng/mL EGF, 20 ng/mL basic FGF, 1% sodium pyruvate, 1% penicillin and streptomycin) similar to our prior reports (57 (link)). Neurospheres were dissociated for 20 minutes with Accutase (Innovative Cell Technologies) at 37°C, viability-quantified with trypan blue utilizing the Countess II (Thermo Fisher Scientific), and plated at 500–1500 viable cells per 100 μL for 24 hours prior to treatment with indicated doses of brigatinib and sitravatinib (Selleckchem). After 7 days, CTG reagent (Promega) was added at 20 μL per well for 30 minutes at 37°C, prior to reading luminescence on the BioTek Synergy H1 (Agilent). Raw values were corrected to untreated (DMSO) control.

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Stackhouse C.T., Anderson J.C., Yue Z., Nguyen T., Eustace N.J., Langford C.P., Wang J., Rowland JR I.V., Xing C., Mikhail F.M., Cui X., Alrefai H., Bash R.E., Lee K.J., Yang E.S., Hjelmeland A.B., Miller C.R., Chen J.Y., Gillespie G.Y, & Willey C.D. (2022). An in vivo model of glioblastoma radiation resistance identifies long noncoding RNAs and targetable kinases. JCI Insight, 7(16), e148717.

Publication 2022

Accutase Countess II brigatinib CTG reagent BioTek Synergy H1

Accutase Brigatinib Cells Cultured media Dmso Luminescence Penicillin Promega Pyruvate Sitravatinib Sodium Streptomycin Trypan blue

Corresponding Organization :

Other organizations : University of Alabama at Birmingham, The Ohio State University, Emory University

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Variable analysis

independent variables

Brigatinib (with indicated doses)
Sitravatinib (with indicated doses)

dependent variables

Cell viability/proliferation (measured by CTG reagent and luminescence)

control variables

PDX media composition (DMEM/F12, 2% B27, 20 ng/mL EGF, 20 ng/mL basic FGF, 1% sodium pyruvate, 1% penicillin and streptomycin)
Neurosphere dissociation protocol (20 minutes with Accutase at 37°C)
Cell plating density (500-1500 viable cells per 100 μL)
Incubation time prior to treatment (24 hours)
CTG reagent addition (20 μL per well for 30 minutes at 37°C)
Luminescence reading on the BioTek Synergy H1

controls

Untreated (DMSO) control

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