Cellular Bioluminescence Based Assay: A High Throughput Method for Combinatorial Drug Screening

Protocollo

1. Bio-luminescence Based Measurement of Cell Viability

1. Coating plates with the extracellular matrix (ECM) mixture (e.g., Matrigel): Add 40 µL of 0.15 mg/mL ECM mixture to each well and incubate the plate for 1 h at 37 °C. Remove the excess ECM mixture and gently rinse once with PBS.
2. Add 100 µL culture medium containing 15,000, 10,000, 8,000, 6,000, 4,000, 2,000, 1,000, and 500 XG387-Luc cells together with 100 µL blank medium as control into each well for 6 replicates in a 96-well optical bottom plate and culture the cells overnight at 37 °C.
3. Remove the supernatant, add 50 µL culture medium containing 150 ng/µL D-luciferin into each well and incubate the cells for 5 min at 37 °C.
4. Take images of the cellular bio-luminescence in the plate using the IVIS spectrum imaging system. Use the built-in software to create multiple circular areas of the region of interest (ROI) and quantify the cellular bio-luminescence.

2. Temozolomide Treatment and Combination Screening

1. Precoat four 96-well plates as described above in step 1, prior to the treatment.
2. Seed XG387-Luc cells at a density of 1,000 cells in 100 µL culture medium into each well of a 96-well optical bottom plate and culture the cells overnight.
3. Prepare temozolomide and the targeted agents from the stock solution in advance. Prepare a concentration series composed of 800 µM, 600 µM, 400 µM, 300 µM, 200 µM, 100 µM, and 50 µM temozolomide in culture medium for the single-agent treatment. Dilute temozolomide and the targeted agents in stock solution in the culture medium, respectively, to obtain final concentrations of 200 µM and 2 µM for combination drug screening (Materials list).
4. Remove the culture medium when most of the GSCs adhere to the bottom of the plates; add the above-prepared medium containing temozolomide into each well for three technical replicates per treatment.
5. To treat Temozolomide and to screen the drug combinations remove the blank medium and add the above-prepared medium containing either 200 µM temozolomide, or 2 µM targeted agent, or a combination of both into each well for three technical replicates per treatment.
6. Incubate all plates at 37 °C, 5% CO₂ for 3 days.
7. Remove the drug-containing medium, add 50 µL blank medium containing 150 ng/µL D-luciferin into each well and incubate the cells for 5 min at 37 °C.
8. Take images of the cellular bio-luminescence in the plate using the IVIS spectrum imaging system. Use the built-in software to create multiple circular ROIs and quantify the cellular bio-luminescence.

Materiali

Name	Company	Catalog Number	Comments
B-27	Gibco	17504-044	50X
EGF	Gibco	PHG0313	20 ng/ml
FGF	Gibco	PHG0263	20 ng/ml
Gluta Max	Gibco	35050061	100X
Neurobasal	Gibco	21103049	1X
Penicillin-Streptomycin	HyClone	SV30010	P: 10,000 units/ml S: 10,000 ug/ml
Sodium Pyruvate	Gibco	2088876	100 mM
ABT-737	MCE		Selective and BH3 mimetic Bcl-2 Bcl-xL and Bclw inhibitor
Adavosertib (MK-1775)	MCE		Wee1 inhibitor
Axitinib	MCE		Multi-targeted tyrosine kinase inhibitor
AZD5991	MCE		Mcl-1 inhibitor
A 83-01	MCE		Potent inhibitor of TGF-β type I receptor ALK5 kinase
CGP57380	Selleck		Potent MNK1 inhibitor
Dactolisib (BEZ235)	Selleck		Dual ATPcompetitive PI3K and mTOR inhibitor
Dasatinib	MCE		Dual Bcr-Abl and Src family tyrosine kinase inhibitor
Erlotinib	MCE		EGFR tyrosine kinase inhibitor
Gefitinib	MCE		EGFR tyrosine kinase inhibitor
Linifanib	MCE		Multi-target inhibitor of VEGFR and PDGFR family
Masitinib	MCE		Inhibitor of c-Kit
ML141	Selleck		Non-competitive inhibitor of Cdc42 GTPase
OSI-930	MCE		Multi-target inhibitor of Kit, KDR and CSF-1R
Palbociclib	MCE		Selective CDK4 and CDK6 inhibitor
SB 202190	MCE		Selective p38 MAP kinase inhibitor
Sepantronium bromide (YM-155)	MCE		Survivin inhibitor
TCS 359	Selleck		Potent FLT3 inhibitor
UMI-77	MCE		Selective Mcl-1 inhibitor
4-Hydroxytamoxifen(Afimoxifene)	Selleck		Selective estrogen receptor (ER) modulator