## Sterile Technique, Pipetting, Dilutions and Graphing Results

Paper details:

You will need three graphs, each with proper error bars, for differentiation, proliferation and viability. Excel sheets have been supplied, and can be downloaded from this Top Hat folder. Instructions are given with the differentiation results, so start there and work your way through that one before you go on to the other two sheets. There is also a file with information on writing a figure caption.

It is crucial that you create proper error bars on your graphs in order to interpret your data correctly. The default in Excel is to put the same amount of error on each data point in a series, but if you do this, you will not be able to interpret your data correctly. This would severely compromise both your Results and your Discussion! Error bars should indicate standard error above and below the mean. There is a step by step guide on the differentiation sheet. (If you are using a Mac, there will be some differences.)

When you look at your error bars – and you do need to – look first to see where they overlap and where they do not. If you can draw a straight line horizontally through the error bars, you can conclude that no statistical difference was found, within error. If they do not overlap, you can conclude that statistical difference was observed, within error.

(However, whether or not you are able to draw statistical conclusions from data depends of the number of replicates used. If there are fewer than 6 replicates – 2 independent data points from 3 independent experiments – you cannot reliably use error bars to draw conclusions regarding statistical significance. This is the case for this project, because whereas we have 4 data points for each concentration for each point in time, these were not generated by three independent experiments. For the purposes of this lab, I want to make you aware of this, and if you can comfortably work this into your analysis, please do. However, it is not necessary as long as you understand this. You may want to describe what you see as possible trends that could be confirmed or rejected if you were to repeat the experiment enough times.)

Once your visuals are complete (don’t be afraid to ask!), you can begin the text portion of the Results section. The text is likely to be very brief, because it is a summary, not a point by point descriiption. Try not to describe in words what can already be seen in the graph, but rather look for trends in the data, revealed by the graph. Is there an increased or decreased response over time, for one or more concentrations? Do you see a peak anywhere? A plateau?How do the responses compare to control (0 ng/ml)? (Do not include any interpretations of the biological significance of the data, because that belongs in the Discussion.)

Always introduce the figure in the text of the Results before you show it to your reader. For example, Figure 1 should be inserted as soon as is feasible after noting that: “A general trend showing dose-dependent differentiation was observed (Fig.1).” The text and the visuals should form a coherent presentation of the data.

NGF Calculations
Congratulations! You made it through that part! This part is easier. We need to add NGF.

Chemical messengers such as NGF are present in the body in concentrations that are measured in ranges so low as to be measured in pg/ml (pg = picograms = 10-12 g/ml). If we re-constituted these from powder with too much solvent, they would no longer be stable and this would compromise our experiments. Therefore, we re-constitute and freeze the NGF at a higher concentration, then make more dilute solutions just before adding it to the cultures. By the time we have diluted to this point, we are adding very low volumes. For the differentiation experiments, we never add more than 125 µl to volumes of more than 20.8 ml (2 plates for each concentration with 10 ml each X 1.04; this is less than 0.01% of the total volume). This small volume will not significantly change the volume of other components, and it could introduce error if we try to measure other components to degrees of accuracy not allowed by our equipment. Therefore, although we determine the volume of NGF that will be added for each pair of plates, we can do that last, after we have done all our other calculations, because these will not be considered when we do our other calculations.

Because the content of each plate will be the same but for the NGF, you can make one mixture for all plates. Before you add NGF, you can aliquot into 4 separate tubes, one for each concentration (including 0, the control), then add the NGF, then plate the cells. Use the formulae below to determine how much NGF will be added to each treatment condition, i.e. each one of the four separate tubes. (We will only do calculations for the differentiation part of the experiment; the calculations are the same for proliferation and viability , but with a different number of plates.) The NGF stock will be at a concentration of 10,000 ng/ml.

stock concentration/final concentration desired = dilution factor
final volume/dilution factor = volume of stock to add

Study this example before you begin.

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