Following on from the first assignment (microbiome talk, 30%) and the second task (liquid biopsy piece, 30%), the third assessment for MB1080 picks up on the data handling component of the module (40%, total).
This data handling component is actually comprised of a number of different elements:
(1) Three online assessments based on the use of the R package for statistical analysis (5% each, 15% total). Two of these were shared in common with students on BS1070, and should already have been completed. The third is linked to the current task and should be submitted online
The second and third element (10% and 15%, respectively) put you in the position of a researcher conducting experiments on cell adhesion in atherosclerosis. The deadline to complete these, is rather further off, but we have provided you with the necessary information now, in order that you understand the context of the third Stats assignment. For the record, the other submissions will be:
(2) Undertaking the second part, some calculations regarding solutions and cells to be used in the cell adhesion experiment, i.e. the types we are practicing in some of the MB1080 tutorials. Your results on this part of the task must be submitted online in the format of a Blackboard quiz. We will need some evidence of your calculations, see red text below. On or before you will need to submit:
Cell adhesion experimental summary and results data
Endothelial cells form a single cell layer throughout the vasculature and form the innermost layer of the vessel wall. Here they come into direct contact with the blood, including its cellular components. Under normal physiological conditions endothelial cells have anti-inflammatory and anti-coagulant properties. However, when the endothelium becomes damaged or activated in response to many different cardiovascular risk factors, the endothelial cells change in phenotype and secrete pro-inflammatory cytokines and upregulate cell adhesion proteins. These adhesion proteins are responsible for binding blood monocytes and facilitate their transendothelial migration (Figure 1). Once within the subendothelial space the monocytes differentiate into macrophages and take up lipids, forming foam cells and leading to a “fatty streak” within the vessel wall. This is the first stage of atherosclerosis.
Figure 1. Monocyte adhesion and transmigration. Monocytes roll across the endothelial surface and can bind to adhesion molecules via integrins that are expressed on their cell surface. Binding facilitates migration across the endothelium into the subendothelial space.
One of the most common diseases to result from atherosclerosis is coronary artery disease (CAD). Whilst there are many risk factors (smoking, high cholesterol, hypertension) that contribute to the risk of developing CAD, genetic factors play a key role. In order to understand the contribution of genetic variation researchers around the world have conducted large-scale genome-wide association studies. This has led to the discovery of certain genetic variants, often called SNPs, that increase risk of CAD. One such SNP in “Gene X” is thought to increase risk by upregulating the adhesion molecule ICAM-1 and increasing cell adhesion.
To test whether the genotype of this SNP can influence cell adhesion we can perform a monocyte adhesion assay using endothelial cells grown in the laboratory. This assay involves growing the endothelial cells from individuals of the three possible genotypes (termed AA, Aa, aa) in a plastic cell culture vessel. For each genotype the cells are then moved into the cell culture vessel. The cells are then incubated with monocyte cells (THP1) that have been labelled with a fluorescent dye (Calcineurin) either in normal conditions or after the addition of the pro-inflammatory cytokine TNFα. After incubation, non-adherent THP1 cells are removed by gentle washing. The amount of bound THP1 cells is then measured in each well using a fluorescence spectrophotometer to measure the amount of fluorescence emitted by the labelled THP1 cells. The more cells that are adherent, the greater the fluorescent signal.
Methods (calculations in Task 2 relate to this section)
Endothelial cell preparation
Before the assay is conducted the endothelial cells are cultured in plastic cell culture dishes (Figure 2). They grow adhered to the bottom of the dish within a liquid Endothelial Cell Growth Medium containing growth factors and supplements for optimal growth.
Figure 2: Cell culture dish with liquid medium containing cells Cells grow adhered to the bottom of the dish. Cells are incubated at 37˚C in a humidified atmosphere of 5% CO2 .
Supplement Mix Components Final Concentration Stock Concentration Volume of stock added
Fetal Bovine Serum 2% 100%
Endothelial Cell Growth Supplement 4 μg/ml 1 mg/ml 2ml
Epidermal Growth Factor 1 μg/ml 50μl
Basic Fibroblast Growth Factor 2 μg/ml 250μl
Heparin 90 μg/ml 72 mg/ml 625μl
Hydrocortisone 1 μg/ml 667μl
Table 1: Components added to make the Endothelial Cell Growth Medium
Q1. You wish to prepare a total volume of the 500 ml of the Endothelial Cell Growth Medium. Calculate the missing values to complete Table 1.
a) What volume of stock Fetal Bovine Serum needs to be added? (2 marks)
b) What will the final concentration of Epidermal Growth Factor? (2 marks)
c) What will the final concentration of Basic Fibroblast Growth Factor? (2 marks)
d) What is the stock concentration of hydrocortisone? (2 marks)
Removing the cells from the dish using trypsinisation
Once the cells are sufficiently grown (confluent), they need to be removed from the culture dish and counted in preparation for the cell adhesion experiments. The liquid medium is removed from the dish and the cells are washed in pre-warmed (37˚C) 1x phosphate buffered saline (PBS). 1.5ml of 1x Trypsin-EDTA solution is added to the cells and they are incubated for 2 minutes at 37˚C to detach the cells from the dish. The trypsin is then deactivated by further addition of 5% fetal bovine serum (FBS) in PBS.
Q2. PBS for the experiments (“1x solution”) contains:
NaCl (Mr = 58.44) 137 mmol/l
KCl (Mr = 74.55) 2.7 mmol/l
Na2HPO4 (Mr = 141.96) 10 mmol/l
KH2PO4 (Mr = 136.09) 1.8 mmol/l
To facilitate these experiments you make up a 1 litre stock of 5x PBS.
(a) How much NaCl will you need to add to this 5x PBS stock (2 marks)
(b) How much KCl will you need to add? (2 marks)
(c) How much Na2HPO4 will you need to add? (2 marks)
(d) How much KH2PO4 will you need to add? (2 marks)
(e) The 5X PBS solution needs to be adjusted to a final pH of 7.4. In practical terms, how would you make up this solution? (1 mark)
Q3. The Trypsin-EDTA solution used in the experiment would contain 0.4% trypsin (w/v) and
0.03% EDTA (w/v). It is provided to you as a 10x stock solution.
(a) EDTA has a molecular weight of 292.24. What is the molarity of the EDTA in the 10X stock solution? (3 marks)
(b) What is the total final amount (in mg) of Trypsin which is added to the cells? (2 marks)
Q4. Following a 2 minute incubation, FBS solution is then added to the cells to deactivate the trypsin enzyme. The solution comprises 10% FBS, made up in 1x PBS. You decide to make up 50 ml of this solution using your 10 x PBS stock, and a 100% FBS stock.
(a) How much 10 x PBS will you need to use to make the 50 ml solution? (1 mark)
(b) How much 100% stock FBS would you need? (2 marks)
The cells are then centrifuged and resuspended in 1 ml of media and counted using a haemocytometer (Figure 3).
Figure 3: A haemocytometer grid for counting cells. The number of cells in the four outer squares (one is shown in red on the figure) are counted and the result is used to determine the total number of cells in the 1 ml volume. The red square holds 0.1 µl of cell suspension.
For the purposes of the cell adhesion experiment, the cells are then diluted in media such that a 200 µl volume contains 9 x 103 cells. This volume is then added per well for the cell adhesion assay. In total you will test 40 wells of endothelial cells from each genotype for each experimental condition (with or without TNFα).
1) On one occasion, a haemocytometer count of four 0.1 µl volume squares on the grid, are shown to contain a total of 320 cells. What is the total number of cells in the 1 ml volume? (3 marks)
2) On another occasion, a haemocytometer count reveals that your initial culture has 5.4 x 106 cells per ml and will need to be diluted to use in your cell adhesion assay. Recognizing that you should always transfer a minimum of 50 μl of cells between tubes when carrying out a dilution, you decide to go via two intermediate solutions, at 1.8 x106 cells/ml and 1.8 x 105 cells/ml, respectively, and to make 1 ml of each.
a) How many μl of the original 5.4 x 106 cells/ml culture will you need to transfer to make 1 ml of the 1.8 x 106 cells/ml intermediate? (2 marks)
b) How many μl of the 1.8 x 106 cells/ml intermediate will you need to transfer to make 1 ml of the 1.8 x105 cells/ml? (2 marks)
c) How many μl of the 1.8×105 cells/ml intermediate will you need to transfer to make the 200μl solution added to a cell culture well? (2 marks)
Part B – Methods (continued)
Wells in the cell culture vessel are seeded with endothelial cells from each of the three genotypes per experiment (Figure 4). One experiment is testing the difference in adhesion of monocytes between the genotype groups under normal conditions. These are left in normal growth media for 24hrs before the assay. The second experiment is performing the same test but with additional stimulation of the cells with TNFα. To the cells in this experiment TNFα is added (10ng/ml) 24hrs before the assay.
Figure 4 Simplified scheme of cell adhesion plate layout. Endothelial cells are seeded into the wells of the cell culture dish at 9×103 cells/well in 200µl of media.
After 24 hours, THP-1 cells (1 x 106cells/ml) are labelled with 5μM Calcineurin and 100μl of the THP-1 cell suspension is added to the wells containing the endothelial cells and incubated at 37°C for 90 minutes (Figure 5). Following the incubation, the cells ware washed four times with pre-warmed ECGM media. The number of adherent THP-1 cells is read as the amount of total fluorescence per well using a NovoStar (BMG LabTech, Bucks, UK) fluorescence spectrophotometer at an excitation wavelength of 490nm and emission wavelength of 510nm.
Figure 5 Simplified procedure for the adhesion assay. A) Fluorescently labelled THP1 monocytes (green) are added to each well of endothelial cells (orange) and incubated. B) Only adherent THP1 cells remain after gentle washing.
In this study you are comparing three different genotypes to test if these genotype (AA, Aa and aa) influence the endothelial cells ability to bind to monocytes. To generate reliable data you will have repeated the experiment a number of times. The data generated includes 40 repeats.
In the analysis of this experiment you are asking two questions:
(i) Does the genotype have an effect on the amount of bound THP1 cells in normal physiological conditions?
(ii) Does the genotype have an effect on the amount of bound THP1 cells after prior stimulation with the cytokine TNFα?
Results (calculations in this section relate to Task 1, and will inform your answer to Task 3)
The results of the experiment will be made available during and after your session with Dr Mallon on You will need to evaluate these results for Task 1 (submitted by nd reflect on the findings to inform your title and abstract (Task 3, submitted ).
The information you require for the calculations for Task 2 are included in this document. You could start looking at these immediately. However, we will be releasing some more “how to” videos modelling these, and there will be some practice questions of similar format to complete in preparation for the Worksessions on . The links to submit your results (as a Blackboard quiz) and the evidence of your workings [format to be confirmed] will be made available after the optional data handling session o.
Version 1.2, 22nd Feb 2021 (amended text in Red)