Results: Need description of the image as this: Observations about insulin granule distribution described as 1 text – see rubric Need: figure legend for this pic. And also observations in the image....

report


Results: Need description of the image as this: Observations about insulin granule distribution described as 1 text – see rubric Need: figure legend for this pic. And also observations in the image. Properly presented fluorescence microscope images and observations from 1 selected fluorescently-stained microscope slide provided please see rubric (need it detailed as its 3 marks) Observations about stimulation conditions described in the text – please see rubric (need it detailed as its 3 marks) Discussion: Please see rubric and follow the each criteria asked and make sure each one is addressed- please see the marks to know how much to write for each. GSIS 2.8mM glucose 16.7mM glucose 16.7mM glucose + X16.7mM glucose + Y1.11598999999999986.946710000000000410.332292.2915399999999999Conditions [Insulin] ng/ml Insulin ELISA Standards 12.86.43.21.60.80.40.200.850000000000000090.42600000000000010.236000000000000020.134000000000000017.0000000000000021E-24.9000000000000009E-23.500000000000001E-20.05[Insulin] ng/ml Background Corrected Absorbance Report assessment guidelines The report has a three-page limit; any text exceeding this limit will not be marked. References and Appendix are not included in this page limit. The minimum font size is 11. The minimum margins are 2cm. A penalty of 20% will be incurred if these limits are exceeded. The report should be written as a formal scientific communication. It is expected that the report will consist of the following sections: 1. Introduction and aims 2. Materials and methods 3. Results 4. Discussion and concluding remarks The following sections are not included in the page limit: 5. References 6. Appendix – any information that you think should be included in the report can be presented here and it can be referred throughout the report. All figures and tables should have an appropriate title and legend, placed either below or above, respectively, with sufficient information for a reader to understand how the results were obtained, including important information such as concentrations and duration of stimulation conditions etc. If in doubt look at a peer reviewed article that has used a similar experiment or assay for the best example. Marking Scheme Include your group’s student IDs in the file-name and front page of your report. INTRODUCTION (/10) Criteria Mark What are β-cells? Why are they important? 2 How do β-cells function? What stimulates them and how can other substances perturb the normal responses to stimuli? 2 How does β-cell function change from healthy to insulin resistance and finally to Type 2 Diabetes 2 Describe how you observed the function of β-cells in these practicals? Include explaining the basic principal of a sandwich ELISA 2 Aims of the experiments 2 MATERIALS AND METHODS (/2) Criteria Mark Properly reference lab manual, and any variations to the protocol included 1 Correct formatting (third person, past tense) 1 RESULTS (/16) Criteria Mark Practical 1 data: Fluorescence microscopy images: properly presented in a panel, including individual colour and bright-field channels and an overlay of blue and green colour channels, with appropriate labelling and scale bars 4 Practical 1 data: Observations about insulin granule distribution described as text 1 Practical 1 data: Properly presented fluorescence microscope images and 4 observations from 3 1 selected fluorescently-stained microscope slide provided Practical 3 data: ELISA standard curve and GSIS data presented as a column graph with adequate figure title and legends 4 Practical 3 data: Observations about stimulation conditions described in the text 3 DISCUSSION (/14) Criteria Mark Overview of the experiments. (What did you aim to do? What were your main findings?) 4 Discuss the distribution of insulin staining in the β-cells and compare to literature. 2 Discuss the differences between paired fluorescent microscopy samples. *Discuss the visual observations from one provided microscopy slide 2 *Discuss how your ELISA results compare to the class average (class average 4 to be provided on Canvas) Discuss what substances X and Y, from Practical 3, could be based on your results. Use references. Concluding remark, summarise your findings and what was learnt. 2 REFERENCES (/2) Criteria Mark Five to ten journal articles used with consistent referencing style 2 Any referencing style is fine so long as it is consistent throughout the report (e.g. do not refer to an article as [2] and then (Smith et al., 2009) elsewhere). Sheet1 Average Stimulation Conditions[Insulin] ng/mL 2.8 mM glucose0.9266 16.7 mM glucose9.9448 16.7 mM glucose + X18.2125 16.7 mM glucose + Y1.4875 GSIS: TUESDAY Class Average 2.8 mM glucose16.7 mM glucose16.7 mM glucose + X16.7 mM glucose + Y0.926605715495730459.944776802240603818.2125129593623291.4875478080881237Conditions [Insulin] ng/mL Practical class 1: Immuno-fluorescent staining of MIN6 beta-cells General Outline Today you will visualise the distribution of insulin granules in mouse pancreatic β-cells (MIN6) using immuno-fluorescent staining. Each group will have one glass coverslip on which cells have been settled and fixed onto. Following staining and mounting of the coverslip onto glass slides, each group will obtain images from two different regions for their reports using FLOID microscopes. Additionally, you will obtain multi-colour fluorescent images from 3 microscope slides, stained with 3 fluorophores, filling in the worksheet to describe features observed in each tissue or cell sample. Aims Students will be working in groups of four to: • Gain experience with immuno-fluorescent staining of mouse pancreatic β-cells • Gain experience using FLOID microscopes to visualize immuno-fluorescent staining • Learn the proper formatting of microscopy images • Present research data in a scientific report Flow chart Before class create a summary flow chart of the experiment to be carried out. Please have this checked by your demonstrator before beginning your experiment Protocol: Immuno-fluorescent staining Please ensure before you start you have the following: • One 3.5cm cell culture dish containing a glass coverslip with cells • Wash Buffer • Secondary antibody solution TO BE KEPT IN THE DARK • Mounting media containing DAPI TO BE KEPT IN THE DARK • Nail polish for sealing (see demonstrator for use) • One glass slide (YOU MUST CLEARLY LABEL WITH PENCIL): 1. Date 2. Group initials 3. Target of the primary stain (i.e. insulin) 4. Wavelength of the fluorophores used to stain (i.e. 488 for the 488ηm fluorophore) 2 Steps 1 - 7 have already been done for you. Please continue from step 8. 1. Wash: Remove cell culture media and wash twice with 500µL of PBS 2. Fix: Crosslink proteins to preserve cellular structures by using 500µL of 4% PFA for 20 mins 3. Wash: Remove PFA and wash twice with 500µL Wash Buffer (0.1% BSA in PBS with 0.01% Sodium Azide) 4. Permeabilise: After fixation, cell membranes are permeabilised by incubating the cells with 0.01% SDS for 5 minutes. This will allow the primary and secondary antibodies to access the intracellular space. 5. Block: Prevent non-specific staining by using 500µL of Blocking Buffer (DakoTM) for 1 hour 6. Remove blocking buffer, do not wash 7. Primary antibody incubation: Add 100µL pre-diluted guinea pig anti-insulin and incubate overnight at 4°C. Primary antibody has been removed from the cells and wash buffer has been added. 8. Wash coverslips 2 times: Gently remove the wash buffer from on top of the cells by SLOWLY tilting the culture dish 45° and using a p1000 pipette to SLOWLY aspirate (remove and discard) the liquid that has accumulated at the bottom of the well. Add 2mL wash buffer in the same manner by SLOWLY tilting the plate at 45° and SLOWLY adding it to the bottom of the well before SLOWLY setting it horizontally. Gently remove the wash buffer as described earlier. This counts as 2 wash steps. 9. Secondary antibody incubation: SLOWLY add 50µL of secondary antibody (prediluted anti-guinea pig Alexa Fluor 488) onto the coverslips from the corner of the glass as to not disturb the cells on the coverslip. SLOWLY place a square of parafilm over the secondary solution (the surface tension will hold the small amount of solution onto the coverslip). Cover the dish with aluminium foil, and incubate at room temperature for 1 hour in the dark. During the incubation time there will be demonstrations on how to image using the FLOID. FROM THIS POINT ON YOUR SAMPLES MUST BE KEPT IN THE DARK AS MUCH AS POSSIBLE! THIS IS TO PREVENT BLEACHING OF THE SECONDARY FLUOROPHORES 10. Wash coverslips 2 times: SLOWLY Remove parafilm from coverslip and wash as described in step 8, leave 1mL fresh wash buffer on the coverslip. A DEMONSTRATOR WILL COMPLETE STEPS 11 – 13 WITH YOUR GROUP 11. Dry coverslips: Carefully remove coverslips from the culture dish and place it (cell side up) onto paper towels. Let the coverslips dry for ~5 minutes at room temperature, keeping protected from light. 12. Mount coverslips: drop 30µL of mounting media containing DAPI (a blue fluorescent dye that binds to DNA, allowing visualisation of the nuclei of cells) onto each glass slide before carefully lowering the glass coverslip (cell side down) onto the droplet (aiming the center of the coverslip to the droplet). The weight of the coverslip and surface tension of the mounting media will draw out the mounting media to the edges of the coverslip, so it is not necessary to press down on the coverslip and squash the cells. Let the coverslip sit for at least