20.109(S15):Flow cytometry and paper discussion (Day6)

Introduction

We hope that you’ll leave lab today with a sense of accomplishment, after inspecting your raw flow cytometry data and then calculating NHEJ repair values. Unfortunately or excitingly – depending on your perspective – it turns out in scientific research that the hard work is just beginning once the data is quantified! Interpreting the data and drawing (sometimes tentative) conclusions requires deep reading and thinking – a process that shouldn’t be rushed. To help get you started, let’s review a few key elements of DNA repair and of the NHEJ pathway in particular.

Our discussions of NHEJ have primarily been focused on Ku80 and DNA-PKcs, along with a few other key players such as Ligase IV, and will remain so here. However, the schematic below from the review by Grundy et al. highlights that many accessory molecules have their own roles to play. Recall that Ku80, with its dimer partner Ku70, completes the first step of NHEJ repair by binding to DNA double-strand breaks (DSBs). Keep this function in mind as you consider how a Ku80 knockout strain might respond to DNA damage. The Ku 80/70 dimer quickly recruits DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase, to form the DNA-PK complex. Although DNA-PKcs has some binding affinity for DSBs, this affinity is increased by two orders of magnitude by the presence of the Ku dimer, and the kinase activity itself cannot proceed in the absence of Ku. Interestingly, although the kinase activity of DNA-PK is known to be important, it is not certain precisely which phosphorylation events are absolutely required for NHEJ.

NHEJ pathway overview from GJ Grundy et al., "One ring to bring them all—The role of Ku in mammalian non-homologous end joining" in DNA Repair 2014 TBD, in press

The one piece of wet lab work that you will do next time 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, because the stain that we will use contains methanol. (Correction! Our stain contains very little methanol, so 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 recognize 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.

Most of your time today will be spent at the computer, quantifying flow cytometry data. Recall from the M2D5 introduction that we will proceed in three main steps.

First, reporter expression for GFP and BFP alike will be calculated by multiplying percentage of positive cells by fluorescence intensity (FI). We have a choice of whether to use mean, geometric mean, or median fluorescence intensity. Median fluorescence is least susceptible to being influenced by a few outliers, while geometric mean is generally more appropriate for log scale data than arithmetic mean. For normally distributed populations, all three values should be pretty similar. In practice, we have found that while mean and median FI are very different values, after normalization the ultimate NHEJ repair values are quite similar, so we will use the mean value.

The second step is to calculate the ratio of BFP to GFP reporter expression for each sample. The final step is divide the damaged-BFP:GFP ratio by the maximal possible “repair,” namely the intact-BFP:GFP ratio. Convince yourself that this parameter essentially provides the fraction of BFP plasmids repaired.

Protocols

Part 1: Paper discussion

As described in the Day 5 FNT, we will be discussing the Goglia et al. paper in class today.

Technical Background

The paper by Goglia et al. utilizes a fluorescence based DNA repair sensor similar to the one that you are employing in Module 2. There are, however, some important differences in the construction and function of the EJ-RFP (end joining-red fluorescent protein) sensor versus the pMAX-BFP-MCS sensor that was constructed for 20.109. Another paper from the same lab, published in 2013, details the development of the EJ-RFP sensor. You do not need to read the entire paper, but make sure that you understand how the sensor works so that you can fully grasp the high throughput screen that was completed in the Goglia et al. manuscript.

In particular, please read the Introduction and the first two Results sections of the 2013 paper. You will find this background presentation to be helpful for understanding the DNA repair sensors in the Goglia et al. paper.

Discussion Topics

Content

The following questions will guide our in-class discussion; consider them as a starting point rather than a check-list.

Part 2: Flow cytometry analysis

Overview:

• You will begin by looking at images from the instructor samples to learn how to read the flow cytometry plots and summary statistics.
• Next you will peek at your own images and form preliminary expectations about your data set.
• Finally, you will work in Excel to precisely calculate the NHEJ repair value for each of your three conditions (two replicates each).

Protocol:

1. On one of the lab computers, double-click on the FACS server shortcut.
   • Alternatively, on your own computer access 18.159.2.11 directly. Ask your instructors for the username and password.
2. Go to the April 2014 folder, then to Agi Stachowiak. Copy over both the T/R and W/F image sets to your laptop: the filenames begin "analysis-images" and only the dates differ.
3. Copy over just your own day of statistics, unless you really want access to all of the raw data in your back pocket: the .csv filenames begin "analysis-statistics" and only the dates differ.
4. The instructor samples are listed in the table below. From this table, and from the T/R and W/F image sets, try to address the questions below.
   • Background. The scatter data is used – in three steps – to make gate P3, which should consist primarily of live, single cells. From the cells gated in P3, two sub-gates are made that capture all GFP-positive cells ("Green cells" gate) and all BFP-positive cells ("Blue cells" gate). Both singly and doubly positive cells are included in each gate. It is important to read the "% Parent" statistics: these indicate XFP-positive cells as a percentage of all the cells in P3. The "% Total" statistics include debris, aggregates, and clearly dead cells!
   • What percent Green cells are in the mock sample on each day? What about Blue cells?
   • What percent of singly-transfected cells express GFP? Do within-day and cross-day replicates agree well or not?
   • What percent of singly-transfected cells express BFP? Do within-day and cross-day replicates agree well or not?
   • What percent of co-transfected cells express GFP? Express BFP? Comparing the Green and Blue gates to Q1 and Q4, about what percent of cells seem co-transfected, versus expressing just GFP, and expressing just BFP?
   • How is within-day and cross-day replicate agreement for the co-transfected samples? Do the tables below suggest an explanation for why?
   • Does ethanol appear to affect scatter profiles? What about affecting GFP, BFP, or co-expression?
   • What NHEJ repair value do you calculate for Zac's original BFP plasmid, using the first replicate in the W/F instructor data? Try this calculation by hand, using the mean fluorescence intensity. Later, you can include this data as a check on your Excel worksheet. The value you should calculate is 12.8%.Update: Your instructor picked off GFP mean fluorescence instead of BFP mean fluorescence for the intact case! Here is where computers definitely beat manual picking off of data. The correct number is 8.7%.
5. After you understand the instructor data, skim over your 12 sample plots. Can you see apparent differences between K1, K1+401, and xrs6?
6. Now that you have a good conceptual understanding of the data, it's time to crunch some numbers. Open the .csv file and save it as a newly named .xlsx file.
7. Begin by deleting all of the rows except the twelve containing your own dataset.
8. Next delete all of the columns except the few that interest you. Keep in mind that you need to know Green cell and Blue cell gating as a % of the parent gate, P3. Class-wide, you are only required to do your calculations based on mean fluorescence intensity (MFI), to be consistent with Samson lab data. However, you may find it interesting to see whether using median fluorescence intensity gives you the same trends or not. Just a few extra copy-pastes to do both calculations!
9. We recommend that you prepare a new Excel file with your NHEJ equations, and just copy-paste in the appropriate % and MFI data; this approach is a versatile one. Your final worksheet might look similar to the screenshot below.
10. Remember that for each of the twelve wells you should calculate raw reporter expressions and a BFP/GFP normalized value. Then, for each intact/cut pair you can calculate an NHEJ value. In this way, we should have quadruplicate NHEJ values for most repair topology/cell population conditions, which will allow us to do statistical comparisons.

Reference information:

TR instructor samples.

WF instructor samples.

Sample NHEJ calculator screenshot.

You must email your Excel sheet to Shannon (T/R) or Leslie (W/F) before leaving lab today. We instructors will post a summary file for ease of class-wide data analysis by Wednesday evening or Thursday morning.

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