20.109(S14):DNA repair assays(Day6)

Introduction

S14 REVISION IN PROGRESS

The flow cytometry machine has revolutionized biology by allowing researchers to analyze and isolate cells based on their spectral qualities. Both genetic reporters and physical tags may be used to introduce fluorescence into cells. For example, if you have a fluorescently tagged antibody that preferentially binds to a unique cell receptor – or more likely, a set of antibodies that taken together identify a unique cell type – you can isolate a pure sample of this cell type from a complex mixture by using FACS: Fluorescence Activated Cell Sorting. Physical tags less specific than antibodies, such as propidium iodide, are also used in flow cytometry. Propidium iodide is a DNA intercalating agent that cannot pass through viable membranes and thus identifies dead cells. In addition to purification, the flow cytometer can count the number of cells that have a certain spectral quality. Technically, if the machine is used just for counting and not for separating subpopulations of cells, then the procedure is called flow cytometry, while cell sorting or isolation is called FACS. However, because FACS is shorter to say than flow cytometry, many people call anything to do with a flow cytometer FACS, much to the annoyance of pedantic fools like your instructor. So… let's start a revolution, and label flow cytometry FC from here on – even shorter than FACS!

Before there were flow cytometers, there were Coulter counters. Coulter counters are automated cell counting machines developed in the 1950s that count cells as they flow in a liquid stream. In an ingenious conceptual leap, Mack Fulwyler combined the technology of ink jet printers with that of Coulter counters to develop the first flow cytometer. The ink jet printer head works by vibrating a nozzle so that a spray of discrete droplets is formed. Similarly, in a flow cytometer, a liquid suspension of cells is forced at high pressure through a vibrating nozzle to create tiny charged droplets, each containing a single cell. The stream of droplets pass in front of a laser beam, and the scattered light is analyzed by a series of filters and photomultiplier tubes that convert the light signal into electrical impulses. Thus, each cell is "interrogated." In FACS, the spectral qualities of the cell are analyzed nearly instantaneously and compared to user-specified spectral qualities. For example, if you have a mixture of green fluorescent cells and non-fluorescent cells, you can ask the machine to isolate the green cells. If a cell registers as green, an electrical charge deflects the cell to make it fall into a collection chamber. In FC, each cells is interrogated, and documented as fluorescent or not, but then all the cells go to the same waste stream.

Flow cytometer

How FACS works

Cell sorting, or FACS, is technically challenging and most FACS machines are only run by experts. In contrast, regular old biologists and biological engineers are often trained to perform flow cytometry as graduate students. In preparation for an FC experiment, the user must appropriately set voltage, compensation, and gating using control samples; failure to set these parameters correctly will result in GIGO (garbage in, garbage out). The next step of an FC is experiment is to measure the experimental samples on the machine. The final step is to document and analyze the statistics output by the flow cytometer; in our case, this step involves making an additional set of analysis gates. Although these three steps will be done by the teaching faculty, we will describe the first two briefly below. We will cover the final step in the Day 7 introduction.

REVISE You will be using flow cytometry to measure the percentage of cells that are fluorescent. You have lipofected cells with two non-functional EGFP genes. Recombination between these two genes can restore the full length EGFP coding sequence so that cells express EGFP. By measuring the percentage of cells that fluoresce green, you will have some measure of the frequency of homologous recombination within mammalian cells.

Flow cytometers have been around since the 1970s, and while the details have gotten fancier (more colors, faster measurements, etc.), the general principles have remained the same. It is truly a testament to the versatility and utility of flow cytometry that the technology does not look to be supplanted by another approach any time soon.

Protocols

Part 1: Prepare cells for flow cytometry

1. Begin by briefly looking at your cells under the microscope. Do the cells in any wells appear less dense or less healthy than in others? Note down any such observations.
2. Aspirate the media from each well according to the protocol below.
   • As you work, tip the plate down a little to pool the media at the bottom of each well.
   • Place your aspirator at the bottom of a well, and suck up the media. Remove all the liquid, but remove the aspirator promptly after that or you may damage/aspirate some cells.
   • Before moving to the next (non-duplicate) well, dip your aspirator briefly (less than a second!) in ethanol. Then hold the Pasteur pipet up to dry and count to 3.
   • Alternatively, you may use a fresh yellow tip on the end of the Pasteur pipet when changing samples.
3. Gently distribute 0.5 mL of warm PBS to each well, using a 2 mL serological pipet.
   • In other words, don't blast the liquid right at your cells.
4. Repeat the aspiration step, again using the ethanol to clear the pipet between samples.
5. Add 0.25 mL of phenol-red-free trypsin/EDTA to each well, using a P1000.
6. Incubate for about 3 min, until cells are rounding up and starting to come off.
7. Meanwhile, label your pre-chilled flow cytometry tubes according to the tables below.
8. Distribute 0.25 mL of media to each well. You are welcome to use the same pipet tip for each well, as long as you don't touch the tip down into the trypsin/well!
9. One well at a time, follow the protocol below to transfer a uniform cell suspension to the appropriately labeled flow cytometry tube.
   • Pipet the full 0.5 mL of solution up and down about four times. Make sure to vary where your tip is in the well, concentrating on the four "edges" of the circle (if the circle were, you know, a square) to more fully resuspend the sticky cells. Check the first well or two under the microscope to be confident of your technique. You should see very few cells.
   • On the final resuspension, have your partner hand you the filter-topped flow cytometry tube. Press the blue tip gently against the top of the filter, and expel the cells. You should see the liquid go right through; if you feel resistance, ask for help.
   • Each tube should be immediately returned to your ice bucket.

Sample order for each team: K1 intact, K1 intact duplicate, C401 intact, C401 intact duplicate, xrs6 intact, xrs6 intact duplicate, K1 damaged, K1 damaged duplicate, C401 damaged, C401 damaged duplicate, xrs6 damaged, xrs6 damaged duplicate.

Team Color	Tube numbers assigned
Red	1-12
Orange	13-24
Yellow	25-36
Green	37-48
Blue	49-60
Pink	61-72
Purple	73-84
Silver	85-96
White	97-108

Part 2: Plate irradiated cells for inhibitor dose response

For next time

first draft of introduction

Reagent list

write something here or not accessible to edit

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