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This volume details widely used and newer lab protocols for studying hypoxic responses in physiology and diseases. Chapters guide the reader through the application of hypoxic conditions, to the techniques of molecular biology, biochemical, cell biology, genomic,bioinformatic, metabolic, and animal studies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Hypoxia: Methods and Protocols aims to provide a valuable set of tools that can be used to study hypoxia and beyond.

Hypoxia: Methods and Protocols

1. Whole Genome Sequencing-based Discovery of Structural Variants in Glioblastoma

Kazimierz O. Wrzeszczynski, Vanessa Felice, Minita Shah, Sadia Rahman, Anne-Katrin Emde, Vaidehi Jobanputra, Mayu Frank, and Robert B. Darnell

2. Whole Genome DNA Methylation Analysis of Human Glioblastoma using Illumina BeadArrays

Jonathan Serrano and Matija Snuderl

3. Establishing Primary Human Glioblastoma Adherent Cultures from Operative Specimens

Amit D. Gujar, Diane D. Mao, John B. Finlay, and Albert H. Kim

Joshua Frenster and Dimitris Placantonakis

5. Isolation of GBM Stem Cells with Flow Cytometry

Julio Inocencio, Joshua Frenster, and Dimitris Placantonakis

6. Lentiviral Transduction of Primary Human Glioblastoma Cultures

Joshua Frenster, Julio Inocencio, and Dimitris Placantonakis

7. Selective Targeting of CD133-expressing Glioblastoma Stem Cells using Lentiviral Vectors

N. Sumru Bayin and Dimitris G. Placantonakis

8. Intracellular pH Measurements in Glioblastoma Cells using the pH-sensitive Dye BCECF

Nataliya Galifianakis, Dimitris G. Placantonakis, and Mitchell Chesler

9. Induction and Assessment of Hypoxia in Glioblastoma Cells in vitro

Jean-Pierre Gagner, Mirna Lechpammer, and David Zagzag

10. Metabolomic Analysis of Glioma Cells Using Nanoflow Liquid Chromatography-tandem Mass Spectrometry

Jingjing Deng, Guoan Zhang, and Thomas A. Neubert

11. Sample Preparation for Relative Quantitation of Proteins Using Tandem Mass Tags (TMT) and Mass Spectrometry (MS)

Hediye Erdjument-Bromage, Fang-Ke Huang, and Thomas A. Neubert

12. Single-Cell RNA Sequencing of Glioblastoma Cells

Rajeev Sen, Igor Dolgalev, N. Sumru Bayin, Adriana Heguy, Aris Tsirigos, and Dimitris G. Placantonakis

13. Evaluation of Radioresponse and Radiosensitizers in Glioblastoma Organotypic Cultures

N. Sumru Bayin, Lin Ma, Dimitris Placantonakis, and Mary Helen Barcellos-Hoff

14. Orthotopic Patient-derived Glioblastoma Xenografts in Mice

Zhongye Xu, Michael Kader, Rajeev Sen, and Dimitris Placantonakis

15. Bioluminescent In Vivo Imaging of Orthotopic Glioblastoma Xenografts in Mice

Joshua Frenster and Dimitris Placantonakis

16. Evaluation of Vascularity, Blood Perfusion, and Oxygen Tension in Tumor Xenografts with Fluorescent Microscopy

N. Sumru Bayin and Dimitris G. Placantonakis

17. Probing Glioblastoma Tissue Heterogeneity with Laser Capture Microdissection

Jean-Pierre Gagner and David Zagzag

18. Flow Cytometric Identification of Tumor-infiltrating Lymphocytes from Glioblastoma

Karolina Woroniecka, Pakawat Chongsathidkiet, Aladine Elsamadicy, Harrison Farber, Xiuyu Cui, and Peter E. Fecci

19. Modeling Glioma with Human Embryonic Stem Cell-derived Neural Lineages

Aram S. Modrek, Jod Prado, Devin Bready, Joravar Dhaliwal, Danielle Golub, and Dimitris G. Placantonakis