20.109(S16):Data analysis (Day9)

Introduction

The final piece of wet lab work that you will do today is complete your NHEJ inhibitor validation assay. Clonogenic assays of mammalian cells have over a 50-year history, as mentioned in the methods paper by Franken et al. They are useful for assessing the reproductive capacity of cells after irradiation and other types of damage. We will diverge somewhat from the Nature Protocols paper, but it is useful for introducing terms such as the plating efficiency and the surviving fraction. Specifically, we do not need to fix our cells in an independent step; fixing does not appear crucial for short-term staining. Second, we will not use the crystal violet stain, which binds DNA, but instead a Coomassie derivative, which targets proteins. In fact, you may remember Coomassie as the go-to stain for SDS-PAGE. Protein binding by the dye occurs primarily via arginine, as well as other basic and aromatic residues, as described here. We will use a variant of the original Coomassie Brilliant Blue stain called BioSafe Coomassie.

Protocols

Part 1: Paper discussion with Prof. Leona Samson

See M2D7 for guidelines on this rescheduled discussion of the research article by Dietlein et al.

Part 2: Stain irradiated cell colonies

Your irradiated cells have now been growing for 12 days. Time to count colonies!

1. Briefly observe your irradiated cells on the TC microscopes. Are you able to find some colonies? About how many cells are in some of these small dispersed clusters?
2. Take your plate to the main lab for the remaining steps. First, aspirate the media. You don't need to change the yellow tip between samples if you move from highest drug dose to lowest, as that should be the order of least to most colonies.
3. Rinse each well with about 2 mL of PBS.
4. After removing the PBS, use a serological pipet to consistently add 2 mL of Coomassie to each well.
5. Place your plate on the shaker at 80 rpm for about 1 hour.
6. Repeat the PBS rinse.
7. Let the well plate dry for a short time after aspirating the PBS.
   • If you wait a very long time the stain will begin to fade.
8. Finally, count the colonies in each well and document these on today's Discussion page.
   • Do your best to apply a consistent standard for threshold colony size and threshold staining intensity. As long as you are consistent, there is not one right answer as to what constitutes a colony.
   • One of your instructors finds it easiest to count colonies by making a dot with a lab marker as she counts each colony, right on the underside of the 6-well plate at the colony location, and then writing down each decade (10, 20, etc.) on the plate as she reaches that number so she doesn't lose her place.
   • Another one likes taking a picture of her plate (using the zoom of the gel documentation station), printing it, and counting colonies on paper.
9. Whether today or on your own, you should plot your data by surviving fraction for the Module 2 report. If you are feeling ambitious (and your data is appropriate) you can estimate the IC50 for your inhibitor. Does it agree with the published values?

Part 3: Applying statistical analysis to data

You may find averages, measures of spread (standard deviation or 95% confidence intervals), and t-tests useful when you report on class results.

You can practice the steps below using the male and female heights that we collect during pre-lab lecture.

1. Begin by downloading the following Excel file as a framework to carry out the basic statistical manipulations we discussed in lecture. The file is modified from one originally written by Professor Bevin Engelward.
2. Find and plot 95% confidence intervals for the male and female heights.
3. Compare the means of these two populations. At what confidence level (if any) are they different?
   • Would a one-tailed or two-tailed test be more appropriate for this comparison?

Reagent list

• PBS
• Bio-Safe Coomassie Stain (Bio-Rad)
• Mostly your brains!

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