Cell density, culture conditions and oxidative stress

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Chapter 2 Materials and methods

This chapter describes the general materials and methods that apply to much of the work conducted thoughout this thesis. Any chapter specific methods will be described within the relevent chapter. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. All phytochemcials were made up in dimethyl sulfoxide (DMSO)

Cell lines and cell culture

All cell lines were maintained in media supplemented with 10% fetal bovine serum (FBS), 100 units per mL penicillin and 100 µg per mL streptomycin (complete medias) and were grown at 37 oC in a humidified atmosphere of 5% CO2 in air. Cell growth and viability was determined by counting trypan blue stained cells on a hemocytometer. Collection of cells for resuspension at different density cultures and for plating for experiments was conducted using a in a Heraeus Multifuge 1 S-R centrifuge(Buckinghamshire, England.) at 300 x g for 5 mins. All cell lines were treated/exposed to no more than 0.2% DMSO during experiments. Unless described specifically, all cells used for enzymatic assays were lysed in lysis buffer containing complete ethylenediaminetetraacetic acid (ETDA)-free protease inhibitor (Roche, Basel, Switzerland) to inhibit the enzymatic breakdown of cellular proteins. Cells were subjected to three freeze-thaw cycles to release intracellular proteins, then were centrifuged at 16 000 g for 15 min (Eppendorf centrifuge 5415D) (Eppendorf, AG, Hamburg)


SH-SY5Y cells are a human neuroblastoma cell line that was originally sub-cloned from SK-N-SH cells. SH-SY5Y cells are known to be reactive to dopamine beta hydroxylase and are acetylcholinergic, adenosinergic and glutamatergic. SH-SY5Y cells are semi-adherent, with 80% of the culture being adherent and 20% being in suspension. The morphology of SH-SY5Y cells resembles neuronal cells that extend neuritis, although they do form large undifferentiated masses. This cell line is genetically female and the original SK-N-SH cells were established in 1970 from a bone marrow biopsy of a metastatic neuroblastoma site. Within this thesis the SHSY5Y cells are being used as an example of cells that are sensitive to oxidative stress and are not being used for any neuronal cell specific measures.Neuroblastoma derived SH-SY5Y cells (CRL-2266™) were obtained from ATCC
(American Type Culture Collection; Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium/Ham’s F-12 Nutrient Mixture (DMEM/F12) (Invitrogen, Carlsbad, CA.) (cat. no. 11320; Gibco Invitrogen). SH-SY5Y subculturing was carried out as follows:
1. Culture medium was aspirated and discarded.
2. The cell layer was briefly washed with phosphate buffered saline (PBS) that was pre-warmed to 37 oC. 2.0 mL of trypsin (0.25%)-EDTA solution (Invitrogen, Carlsbad, CA.) (cat. no. 25200-056; Gibco Invitrogen) was then added to the cells.
3. Cells were incubated at 37 oC in a humidified atmosphere of 5% CO2 in air to facilitate detachment and dispersal.
4. 8.0 mL of DMEM/F12 medium was added and cells were aspirated gently by pipetting.
5. Cells were then subcultured at a ratio of between 1:20 and 1:50.
For experiments SH-SY5Y cells were seeded at densities of either 2 x 104 cells per well in 96-well plates or 2 x 105 cells per well in 24-well plates in complete DMEM/F12 and allowed to attach for 24 h.

Chapter One: Introduction 
1.1 Functional foods
1.2 Diseases of the 21st century
1.3 What is oxidative stress?
1.3.1 Important oxidants
1.3.2 Sources of oxidant generation in the body and antioxidant protection from them
1.3.3 Specific issues within the brain Ischemic stroke Alzheimer‟s disease Brain aging
1.3.4 Oxidative stress, CVD and cancer
1.4 Health benefits of fruit and vegetables 
1.4.1 Absorption and conjugation of phytochemicals
1.5 Cancer etiology and disease prevention by phytochemicals
1.6 Synergies between phytochemical extracts 
1.7 Cell death 
1.7.1 Apoptotic cell death The process of apoptosis Receptor mediated cell death TNFα receptor signaling Fas Receptor Signaling TRAIL Receptor signaling
1.7.2. Mitochondrial mediated cell death Bcl family of Proteins Bax and Bak Bad, Bim and Bid P53
1.7.3 The caspase cascade Cell receptor mediated caspase activation. Mitochondrial mediated activation of caspases. Substrates of the effector caspases
1.7.4 Autophagy mediated cell death
1.7.5 Necrotic cell death
1.7.5 Necrotic cell death
1.7.6 Lysosomal mediated cell death The role of lysosomes in apoptosis
1.7.7 Endoplasmic reticulum mediated cell death
1.7.8 JNK, the oxidant regulated cell signaling pathway JNK activation by oxidative stress
1.8 Regulation of direct and indirect antioxidant actions
1.8.1 Direct chemical antioxidant capacity
1.8.2 Indirect endogenous enzymes mediated antioxidant action
1.9 Questions
Chapter 2: Materials and methods 
2.1 Cell lines and cell culture
2.1.1 SH-SY5Y.
2.1.2 Jurkat
2.1.3 Transformed embryonic kidney (HEK)
2.1.4 HT-29
2.2 Measurement of antioxidant capacity (FRAP) 
2.2.1 Solutions for FRAP assay
2.2.2 FRAP assay method
2.3 Measurement of antioxidant capacity (ORAC) 
2.3.1 Solutions for ORAC Assay
2.3.2 Methods
2.4 Assessment of cell death
2.5 Measurement of catalase activity
2.6 Catalase and glutathione peroxidase
2.7 Measurement of Trx concentration
2.7.1 Solutions for Trx measurement
2.7.2 Procedure
2.8 Measurement of TrxR activity
2.8.1 Solutions for TrxR assay
2.8.2 Procedure
2.9 Assessment of SOD activity 
2.9.1 Solutions for SOD assay
2.10 Measurement of GSH levels
2.11Assessment of MAPKinase activation
2.12 Data analysis
Chapter 3: Cell density, culture conditions and oxidative stress
3.1 Abstract
3.2 Introduction
3.3 Specific materials and methods 
3.3.1 Cell culture conditions and treatment
3.3.2 Caspase 9 activation
3.3.3 Measurement of ATP levels
3.3.4 Measurement of ROS
3.4 Results
3.4.1. Effect of cell number exposed to H2O2 on cell death
3.4.2. Cell death and caspase activation after long term cell culture density treatments
3.4.3. ATP levels
3.4.4. Catalase activity in long term cell cultures grown at different densities
3.4.5. ROS levels
3.5 Discussion
3.5.1 Density effect on apoptosis and necrosis induced by H2O2
3.5.2 Density effect on catalase levels and ROS levels
Chapter 4: Phytochemical screening
4.1 Abstract
4.2 Introduction
4.3 Phytochemical selection
4.4 Specific methods
4.4.3 Synergy testing
4.5 Results
4.5.1 DMSO effects
4.5.2 Caspase activation
4.5.3 Phytochemical cytotoxicity
4.5.4 Phytochemical mediated cytoprotection
4.5.5 Synergy testing
4.6 Discussion
4.6.1 Protection from H2O2 induced cell death
4.6.2 Cellular toxicity of phytochemical compounds
4.6.3 Synergy screening of phytochemical compounds
4.7 Conclusion 
Chapter 5: Mechanisms of 3,4-Dihydroxybenzoic acid (DHBA) mediated protection from oxidative stress-induced cell death
5.1 Abstract
5.2 Introduction
5.3 Specific methods
5.3.2 Cell culture conditions
5.3.5 Quantification of intracelluar oxidative stress
5.3.6 Caspase activation assay
5.4 Results.
5.4.1 Chemical antioxidant capacity of the DHBAs
5.4.2 Protection from cell death and correlation with antioxidant capacity
5.4.3 Intracellular oxidative stress
5.4.4 Caspase activation
5.4.5 Catalase activity
5.4.6 Catalase and glutathione peroxidase
5.4.7 Determination of Trx concentration
5.4.8 TrxR activity
5.4.9 3,4-DHBA mediated changes in intracellular oxidative stress
5.4.10 3,4-DHBA activation of MAPKinases
5.4 Discussion
5.4.1 Chemical antioxidant capacity
5.4.2 Protection from cell death
5.4.3 Relationship between chemical antioxidant capacity and protection from cell death
5.4.4 Protection from intracellular oxidative stress by pre-treatment with 3,4-DHBA
5.4.5 Prevention of caspase 8 and 9 activation
5.4.6 Catalase activity and its relation to cytoprotection
5.4.7 Induction of oxidative stress by 3,4-DHBA treatment
5.4.8 Potential mechanisms underlying transcriptional events
Chapter 6: Regulation of antioxidant enzymes in multiple cell types by cyanidinderived anthocyanin metabolites 2,4 and 3,4-dihydroxybenzoic acid
6.1 Abstract
6.2 Introduction
6.3 Specific materials and methods 
6.4 Results
6.5. Discussion
6.6 Conclusion 
Chapter Seven: Regulation of antioxidant enzymes in Sprague-Dawley rats by the phytochemical metabolite 3,4-DHBA 
7.1 Abstract
7.2 Introduction
7.3 Specific methods
7.4 Results
7.5. Discussion
7.6 Conclusion 
Chapter 8 Final discussion
8.1 Introduction
8.2. What is the role of cell culture density in assays involving oxidative stress? 
8.3. How effective & representative are phytochemical metabolites vs dietary phytochemicals in vitro assays?
8.4. What are the roles of phytochemical metabolites in the protection from oxidative stress? Endogenous antioxidant enzyme effects vs chemical antioxidant action
8.5. Does the use of phytochemical metabolites in in vitro assays improve their relevance to in vivo trials?
8.6 Future Experiments
8.7 Final conclusion

Phytochemical metabolites and their effects on in vitro and in vivo measures of oxidative stress

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