Evaluate the different approaches that could be used to assess the consequence of oxidative stress in cell models of Parkinson’s disease.

Section A

 

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Answer all parts (a-c) and show examples of your calculations.

 

You are a researcher tasked with the setting up a series of experiments and analysing the resultant data from a study of the effects of an organophosphate compound (OPX) on the growth and viability of the cultured human neuronal tumour cell line SH-SY5Y, as indicated below. You must handle, analyse, present and interpret the data provided, providing evidence of how you have arrived at your conclusions.

 

  • Cells were grown as a mitotic monolayer to approximately 80% confluence then detached using trypsin. After centrifugation of the resultant cell suspension, cell pellets were resuspended in 1 mL growth medium. A volume of 10 µL of this cell suspension was mixed with 90 µL growth medium containing 0.4 % v/v Trypan Blue, after which the cells analysed in a haemocytometer, giving the data shown in Table 1. (30 % marks)
Square number* Total cell count Blue cell count
1 75 3
2 102 8
3 89 6
4 95 5
5 85 7

*Each haemocytometer square has a volume of 10-4 ml. ×

 

Using the data in Table 1, calculate the following parameters and show your working in each case to demonstrate how you arrived at your answer.

 

  • TOTAL cell count/ml =

 

  • VIABLE cell count/ml =

 

  • % VIABILITY =

 

  • For an experiment requiring 750,000 viable cells in 15 ml medium which gives a seeding cell density of 50,000 cells/ml, what volume in µl of the counted cell suspension would be required?

 

  • If the cell suspension at 50,000 viable cells /ml were seeded into 96-well microtiter plates at a volume of 200 µl per well, how many cells per well would there be in each well?

 

 

 

Please limit the word count in the sections below to no more than 250 per section.

 

  • Cells were seeded into a 96-well microtiter plate as described above, then allowed to recover for 24 h in a CO2 At this point the culture medium was removed and replaced with fresh medium containing OPX at 0 – 100 µM, before being assayed for their ability to reduce methyl blue tetrazolium salt. The average absorbance values from 4 independent experiments are shown in Table 2.

 

OPX

concentration (µM)

Average absorbance change at 620 nm in each experiment
Expt 1 Expt 2 Expt 3 Expt 4
0 0.475 0.569 0.610 0.556
6.1 0.502 0.525 0.583 0.561
12.5 0.471 0.515 0.575 0.533
25 0.237 0.331 0.312 0.367
50 0.151 0.175 0.190 0.121
100 0.075 0.080 0.091 0.083

 

Plot a clearly annotated graph to show the average changes in MTT reduction ± SD, in the presence and absence of toxin. Comment on the trends observed, the extent of any changes compared to the control and the potential meaning and limitations of these data. (40% marks)

 

 

  • You have been asked to extend the toxicity study to examine the mechanism of cytotoxicity of OPX in more detail. Explain which approaches you would adopt and why what you would expect to achieve through their use. (30% marks)

 

 

 

Section B

 

Answer any TWO questions. The word limit per answer is 750, excluding figures and references. Only the first 750 words will be assessed. Cite appropriate literature sources and include a properly formatted reference list.

 

  • Evaluate the different approaches that could be used to assess the consequence of oxidative stress in cell models of Parkinson’s disease.

 

 

  1. Outline the immunisation and hybridoma process for producing monoclonal antibodies and discuss how the use of knockout mice has aided monoclonal antibody therapy.

 

 

  • Explain the rationale behind the use of phage display in antibody generation and critically evaluate the steps involved in the phage display of antibody fragments.

 

 

  • Discuss the use of Western blot analysis and immunofluorescence staining as end points for toxicity testing in cellular systems, giving examples of their effective application in toxicity screening or in mechanistic studies of toxicity.

 

 

 

END OF PAPER

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