describes a series of interconnected experiments,

This workbook describes a series of interconnected experiments, the data from which you will be analyzing and presenting as a write up of which constitutes 100% of the module mark. Your write up will also include a review of immunohistochemical techniques that can be used to study molecular and cellular physiology.

Within the report you also need to justify what was done in the experiments and why. The experiments relate to the effects of H2O2 on the neuronal SHSY5Y cell line as studied using the Trypan Blue exclusion assay, the uptake of HRP by the cell line and whether this uptake effects H2O2 toxicity, determined using the XTT assay.

The following methodologies were employed within the experiments: Experiment 1: Effects of H2O2 on viability of SHSY5Y neuronal cells determined by Trypan Blue dye exclusion. (a) 24-well plates were seeded with 5 x 105 cells/well and incubated overnight at 37ºC. (b) A fresh stock of 1 mM H2O2 was prepared in serum free media. A series of five dilutions in serum free medium to determine the effect of the H2O2 on the cells were prepared.

(c) Dilutions of the stock H2O2.stock (30 mM) in serum free medium were prepared. (d) A 24 well plate with SHSY5Y neurons already seeded onto it was treated with 500ul of each H2O2.concentration along with a medium alone control – concentrations were tested in quadruplicate. (e) The plate was incubated for 1 h at 37ºC. (f) Following incubation media was removed from each well, and 100 µl of 4% Trypan Blue was added to each well. (g) Plates were then photographed and the images are displayed for you to analyse. You can count cells that have excluded Trypan Blue (viable cells) plus cells that have taken up the blue dye (dead cells). The ratio of live cells/total cells counted can be used to calculate the percentage viability under each condition. Experiment 2: Uptake of Horseradish Peroxidase by SHSY5Y neuronal cells determined by extraction and peroxidase activity measurement. (a) 6-well plates were seeded with 2 x 106 cells/well and incubated overnight at 37ºC. (b) A total of 3ml of a 0.1 mg/ml solution of HRP in tissue culture medium was added to the top three wells of the plate and incubated overnight. To the other three wells medium alone was added. (c) A 100 mM phosphate extraction/assay buffer comprising 88 mM Na2HPO4,12 mM NaH2PO4, pH 7.4 was prepared. (d) A standard solution of 10 µg/ml HRP was prepared. (e) A TMB peroxidase substrate solution along with stop solution was prepared. (f) 96-well microtiter plates pretreated with 200 µl of 5% Marvel were prepared. (g) A 6 well culture plate seeded with SH-SY5Y neurons was exposed to HRP to allow incorporation overnight. Each well was washed with 1ml serum free medium 3 times. (h) The medium was removed and add 1 ml of 100 mM Phosphate buffer added. Cells were scraped off the plate in this buffer and the suspension transferred to a 1.5 ml Eppendorf tube. (i) The tubes were vortexed for 5 min prior to centrifugation in a microfuge for 5 min at maximum speed. (j) A standard curve of HRP was prepared in 100 mM Phosphate buffer. (k) 50 µl of each standard was added to duplicate wells on a 96 well plate. 50 µl aliquots of each cell extract was added to separate wells in duplicate. (l) 50 µl of TMB substrate was added to each well. (m) After 2 minutes, when a blue colour had developed, 50 µl of stop solution was added to each well. (n) Read Absorbance at 450 nm in a plate reader. Experiment 3: Effects of H2O2 on viability of SHSY5Y neuronal cells pretreated with HRP determined by XTT. (a) 96-well plates were seeded with 1 x 105 cells/well and incubated overnight in the absence or presence of HRP (0.1 mg/ml) at 37ºC. (b) After washing with serum free medium, cells were exposed to doubling dilutions of the H2O2.stock (1 mM) for 1 h. (c) XTT was added and the cells incubated overnight at 37ºC. (d) Absorbance of plates was read at 450 nm in a multiwell plate spectrophotometer. Write-Up Report (3000 words) counts 100% of module mark. Due by 13:00 on Monday 9th December 2020 via Turnitin submission. PLEASE NOTE THIS IS A LEVEL 7 MODULE AND THE REORT NEEDS TO REACH MASTERS STANDARD THROUGHOUT TO OBTAIN A PASS. The assessment for this module is an integrated practical write up (comprises 100% of module assessment), to be written in the style of a journal article. The information and areas covered in depth within the lectures and tutorials are valuable resources for completion of the practical write up. The word-limit for the laboratory report is 3000 words – for submissions over this length penalities of 5% per 300 words over the limit will be applied. Abstract, figure legends and reference list are not included within this word count. The laboratory report should comprise of the standard subsections of a scientific paper, and these are listed below along with indicative word count where applicable: •

Abstract – Overview of what you have done, what you have found and what’s significant (200 words max) • Introduction –

Brief introduction to molecular and cellular physiology relevant to practicals undertaken (800-1000 words). Need to cover literature about endogenous peroxidases and hydrogen peroxide toxicity. Also need to cover literature about horseradish peroxidase, its use as an immunohistochemical marker and the observations about neuronal uptake. •

Note: whilst methods used to acquire the data are provided to you, there is no need to replicate these into the article.

• Results – Full details of the results. Tables and/or graphs of measures, with accompanying legends. Images where appropriate (800-1000 words). All graphs should include appropriate error bars, number of samples assayed and an indication of statistical significance for differences. For experiment 1 include viability counts for each dose of hydrogen peroxide tested and a graph of the resultant dose response curve. For experiment 2 include both the standard curve and calculated sample levels, with an indication of method of sample values calculation. For experiment 3 include dose response curves for control and HRP treated cells. Marks will be given for presentation plus textual explanation of the results presented. FOR ALL GRAPHS PRESENTED PLEASE SUPPLY A TABLE OF THE RAW DATA IN AN APPENDIX (THIS SECTION WILL NOT COUNT TOWARDS THE WORD COUNT) BUT WILL BE USED IN THE MARKING OF THE RESULTS SECTION. •

Discussion – Overview of the success of each experiment.

Discuss the observations and review current literature in the field. (800-1000 words) • Conclusions – Main finding(s) of your study (200 words) •

References – MUST BE in Harvard Format, remember quality of reference sources as well as accuracy of presentation will be assessed. REFERENCES As this is a level 7 report – references should be from a variety of sources. You should include an absolute minimum of 6 references, which must include data papers and at least one reference published within the last 12 months. Please note that quality and relevance rather than quantity will be used in the assessment criteria. The bibliography should be referenced according to the Harvard System. At level 7 the following are essential: – Critical Evaluation – (evaluation of contrasting viewpoints) – Evidence of Wide Reading – Referencluves – Good use of Scientific English