Molecular Cell Biology and Physiology

Data Write-up Assignment

Molecular Cell Biology and Physiology

Clinical and Applied Sciences

Level 7

Semester 1

(10 Credits)

CRN 15456

Cell Biology Measurements

Aim

To determine the effects of hydrogen peroxide (H₂O₂) on cell viability, to determine peroxidase (HRP) uptake into cells and to determine whether HRP uptake alters cellular response to hydrogen peroxide.

The 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.

• 24-well plates were seeded with 5 x 10⁵ cells/well and incubated overnight at 37ºC.
• 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.
• Dilutions of the stock H₂O₂.stock (30 mM) in serum free medium were prepared.
• 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.
• The plate was incubated for 1 h at 37ºC.
• Following incubation media was removed from each well, and 100 µl of 4% Trypan Blue was added to each well.
• Plates were then photographed and the images are displayed on mybeckett 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.

• 6-well plates were seeded with 2 x 10⁶ cells/well and incubated overnight at 37ºC.
• 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.
• A 100 mM phosphate extraction/assay buffer comprising 88 mM Na₂HPO₄,12 mM NaH₂PO₄, pH 7.4 was prepared.
• A standard solution of 10 µg/ml HRP was prepared.
• A TMB peroxidase substrate solution along with stop solution was prepared.
• 96-well microtiter plates pretreated with 200 µl of 5% Marvel were prepared.
• A 6 well culture plate seeded with SH-SY5Y neurons was been exposed to HRP to allow incorporation overnight. Each well was washed with 1ml serum free medium 3 times.
• 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.
• The tubes were vortexed for 5 min prior to centrifugation in a microfuge for 5 min at maximum speed.
• A standard curve of HRP was prepared in 100 mM Phosphate buffer.
• 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.
• 50 µl of TMB substrate was added to each well.
• After 2 minutes, when a blue colour had developed, 50 µl of stop solution was added to each well.
• Read Absorbance at 450 nm in a plate reader. Absorbance data from plate is presented on mybeckett for analysis.

Experiment 3: Effects of H₂O₂ on viability of SHSY5Y neuronal cells pretreated with HRP determined by XTT.

• 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.
• After washing with serum free medium, cells were exposed to doubling dilutions of the H₂O₂.stock (1 mM) for 1 h.
• XTT was added and the cells incubated overnight at 37ºC.
• Absorbance of plates was read at 450 nm in a multiwell plate spectrophotometer.
• Absorbance data from XTT assay is presented on mybeckett for analysis.

Write-Up

Report (3000 words) counts 100% of module mark. Due by 13:00 on Monday 9^th 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.

A useful referencing link:

http://skillsforlearning.leedsbeckett.ac.uk/local/Quote_Unquote.pdf

At level 7 the following are essential:

- Critical Evaluation – (evaluation of contrasting viewpoints)

- Evidence of Wide Reading

- References

- Good use of Scientific English

Student Instructions for Submission of Coursework

This module requires you to submit your work online.

You MUST submit your work through MyBeckett using the link set up by the tutor.  Receipt of your work will be recorded.

Your "Turnitin assignments" in MyBeckett can be set up so that you can check your assignment yourself as you submit it. This checking is done by creating a "Similarity Report".  If this report shows that there are some problems with your work, such as un-cited quotations, you should be able to make corrections and re-submit the work again before the due date. More information about Turnitin is available online here: http://libguides.leedsbeckett.ac.uk/mybeckett/turnitin

Please note: Tutors will follow up any suspected breach of academic integrity found after the submission date as per University policy. Late penalties will apply as per University Regulations.

Particular Instructions to Students

Please read carefully the assessment and grade/marking descriptors overleaf.  Note that descriptors for masters level are more stringent than at undergraduate level

Course Title(s):	MSc Biomedical Sciences, MSc Medical Biochemistry
Module Title:	Molecular cell biology and physiology	Level:	7
Assessment Title:	Laboratory report	Weighting:	100% of module

Classification Grade	(High) Distinction 80-100%	Distinction 70 – 79	Merit 60 - 69%	Pass 50 - 59%	Pass 40 - 49%	Fail 30 - 39%	(Low) Fail 0-29%
Knowledge [The theoretical underpinning of a subject, derived from  collective academic enquiry and experts in the field, including knowledge that is contestable]	Additionally demonstrates originality in knowledge generation by suggesting new theoretical or practical approaches.	Demonstrates a deep understanding and mastery of the knowledge base from a comprehensive  range of theoretical and practice approaches	Demonstrates an understanding of a wide and relevant range of theoretical and practical approaches by utilising them effectively	Demonstrates a good but incomplete range of the relevant theoretical and practical approaches	Demonstrates awareness of a limited range of theory and practice knowledge	Demonstrates a very limited or inappropriate range of theory and practice knowledge	Fails to demonstrate awareness or understanding of the knowledge base
Relevance [Content is clearly related / connected/ significant]	Tightly focused on the title throughout. Consistently addresses all reasonable implications,  nuances and assumptions of the title with breadth and depth	Remains tightly focused on the title. Addresses all reasonable implications nuances and assumptions of the title	Communicates effectively by being directly relevant to the title and learning outcomes. Addresses most implications and assumptions of the title.	Relevant to the title and learning outcomes. Some of the implications of the title addressed	Mainly relevant to the title and learning outcomes but occasionally wanders from the point. Begins to address some of the implications and assumptions of the title.	Little relevant material. Brief for assignment not followed. Only intermittent relevance to the title and learning outcomes, Topic is only superficially addressed.	Very little relevant material. Brief not followed. Irrelevant or patchy connection to title with little or no consideration of the topic.
Application [Utilisation of  outcomes/ learning to a concrete or real life situation]	Demonstrates excellent application of conceptual frameworks, principles and theories, showing critical appreciation, originality and vision in the generation of new insights to apply to/ inform practice	Demonstrates very good use of conceptual frameworks, principles and theories and appropriately challenges received opinion, enabling development and sustaining of coherent argument in the generation of new insights to apply to/ inform practice	Demonstrates good use of conceptual frameworks, principles and theories, showing good ability in the generation of new insights to apply to/ inform practice	Demonstrates appropriate use of relevant conceptual frameworks, principles and theories. Ability generate  new insights to apply to/ inform practice	Demonstrates awareness of conceptual frameworks, principles and theories. Some ability to apply new insights to inform practice	Demonstrates unsatisfactory and inadequate application of conceptual frameworks , principles and theories to practice. Lacks focus.	Lacks application of conceptual frameworks, principles and theories to practice. Displays unsafe practice in written work
Analysis and Evaluation [Demonstration of a systematic examination and reasoned judgement on a range of issues/elements/ materials.]	Exceptional deep, detailed, and critical analysis and evaluation of the material resulting in illuminating, and elegant conclusions that extend the sum of knowledge in the area. Creatively demonstrates new concepts, ideas or theories.	Thorough and consistent critical analysis and synthesis. In depth and appropriate analysis of the material resulting in illuminating conclusions which serve to extend the sum of knowledge in the area. Demonstrates the ability to discover new concepts, ideas or theories through analysis.	Clear evidence of critical analysis and synthesis. Good and appropriate analysis of the material resulting in clear conclusions which serve to add to the sum of knowledge in the area.	Evidence of critical analysis. Adequate and appropriate analysis of the material results in generally clear conclusions, which serve to substantiate , but offers little extension of the sum of knowledge in the area.	Generally adequate and appropriate  analysis of the material that results in some illuminating conclusions but does not expand upon the sum of knowledge in the area.	Analysis of the material is superficial and/or fails to result in clear and/or illuminating conclusions. The sum of knowledge in the area is not expanded, challenged or extended.	Analysis of the material is incomplete and superficial. There are little or no conclusions. The sum of knowledge in the area is not expanded, challenged or extended.
Synthesis [A combination of important points from several sources; infers relationships between sources]	Exceptional work, demonstrating originality, independent thought, synthesis of ideas from credible sources and the generation of innovative views	Demonstrates originality. Distinctive work, demonstrating independent thought, synthesis of ideas and the generation of alternative views.	Elements of originality. Good work showing independent thought, synthesis of ideas and critical engagement with alternative views.	Limited  evidence of originality. Demonstration of evidence of independent thought with some synthesis of ideas and critical engagement with alternative views.	Contains some independent thinking. Beginning to synthesise ideas and engage with alternative views.	No synthesis of ideas Expresses a personal position and is uncritical in its conformity to one or more standard views of the topic	Unable to combine evidence or others’ views to create a new perspective of the topic
Structured Argument [Systematic presentation of academic debate/s]	Highly accomplished, original   arguments are presented making creative use of a mode of argument and theoretical model/s to provide an intelligent structure.	Arguments are reliable and presented coherently, making creative use of appropriate mode of argument and/or theoretical model/s to provide an intelligent structure. There is originality in the arguments  presented	Arguments generally clear and coherent, and logically constructed. Uses an appropriate mode of argument   and/or theoretical model/s. There are elements of originality in the arguments  presented	Arguments lack logic and coherence at times. Uses an appropriate mode of argument and/or theoretical model/s.	Arguments lack logic and coherence at times. Generally uses an appropriate mode of argument and /or theoretical model/s.	Very limited coherence and logic. Fails to make adequate use of an appropriate mode of argument and/or theoretical model/s.	The work is confusing. Arguments lack clarity. There is no evidence of attempts to use appropriate model/ mode of arguments
Presentation and referencing	Presentation is polished with a creative approach to the topic. A fluent academic writing style with accurate spelling and syntax. Thoughts and ideas expressed clearly. Appropriate and complete references from a diverse range of sources.	Presentation polished with an imaginative approach to the topic. A fluent academic writing style with accurate spelling and syntax. Thoughts and ideas expressed clearly. Appropriate and complete references from a range of sources.	Presentation logically structured. Very well written with correct spelling and syntax. Style is lucid with appropriate format. Appropriate , complete and consistent references which may contain minor inaccuracies.	Presentation satisfactory and meaning apparent but language not always fluent. Spelling and syntax satisfactory. Some in-consistencies in referencing but generally acceptable.	Presentation is logical but has minor lapses from standard syntax and spelling. Generally lucid. Acceptable format. Some in-consistencies in referencing but generally acceptable.	Presentation disorganised. Rather poorly written with significant deficiencies in expression, and format. Significant errors in referencing which is incomplete.	Presentation  very disorganised. Very poorly written with significant deficiencies in expression and format References incomplete and unacceptable