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

Published: April 30, 2023

Abstract

Source: Hu, Z. et al. A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells. J. Vis. Exp. (2021)

In this video, we describe a method highlighting the utility of bioluminescence assay for sensitive and rapid screening of drugs. These drugs can be tested alone or in combination to assess the synergism in their mode of action.

Protocol

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.

Disclosures

The authors have nothing to disclose.

Materials

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

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Cite This Article
Cellular Bioluminescence Based Assay: A High Throughput Method for Combinatorial Drug Screening. J. Vis. Exp. (Pending Publication), e20614, doi: (2023).

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