20.109(F10): Mod 1 Day 8 FACS analysis
FACS analysis
Introduction
Today 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. We will use the BioAnalyzer from Agilent Technologies to measure the fluorescence. This equipment has similarities and differences to traditional FACS and flow cytometry techniques.
Traditional FACS and Flow Cytometry
FACS stands for "Fluorescence Activated Cell Sorting." The FACS machine has revolutionized biology by allowing researchers to isolate cells based on their spectral qualities. For example, if you have a fluorescently tagged antibody that preferentially binds to a certain cell type, you can isolate a pure sample of this cell type from a complex mixture by using a FACS machine. In addition to purification, the FACS machine can count the number of cells that have a certain spectral quality. If a FACS machine is used just for counting and not for separating subpopulations of cells, then the procedure is called "flow cytometry." FACS is technically challenging and most FACS machines are only run by experts. In contrast, biologists are often trained to perform flow cytometry in order to analyze the proportion of their sample that has particular spectral qualities.
Before there were FACS machines, 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 FACS machine. The ink jet printer head works by vibrating a nozzle so that a spray of discrete droplets is formed. Similarly, in a FACS machine, 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."
For FACS, the spectral qualities of the cell are analyzed nearly instantaneously and compared to your desired 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.
The BioAnalyzer
This "in lab' instrument works in ways that are similar to the flow cytometer but uses microfluidics, vacuum pumps, and a disposable chip to present the cells one by one to a detector that measures red and blue fluorescence. A very useful animation of the microfluidics is found here. In short,
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Six samples can be loaded on a single chip for analysis
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Each sample (shown in yellow) is pumped though a maze of microcapillary tubes to meet with a viscous liquid (shown in green).
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The difference in viscosity between the samples squeezes the cells so they pass one by one past an optical detector
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allowing each cell to be queried for "redness" and "blueness."
Sensibly, the blue detector will measure GFP fluoresence from our cells. To know we are looking at intact, living cells and not fluorescent cell debris, we will also be staining the cells with a special red dye. CellTracker Red can freely diffuse through cellular membranes, and then inside the cytoplasm it is cleaved to an impermeable derivative. Thus living cells with intact membranes retain the dye and fluoresce red, while the disrupted membranes on dead cells do not. By comparing the number of red cells to the number of red and "blue" (i.e. green from GFP) cells in each sample, we'll know the frequency of recombination between the delta5 and the delta3 constructs, as well as the number of frequency of green cells in our control samples.
Protocol
Preparing your cells for the BioAnalyzer
The following protocol should be performed in the sterile hoods unless otherwise indicated.
Part 1: Labeling Cells for Viability
Part 2: Collecting and Counting Cells
Data collection from transfected cells
For next time
- Please submit your finished "memo." This assignment is due by 11:00 a.m. on the day you have lab. Please turn in your memo electronically by uploading it to the Stellar website that is associated with our class. It is important that you name your file according to this convention: Firstinitial_Lastname_LabSection_assignment.doc, for example: S_Hockfield_TR_memo.doc There will be a 1/3 letter grade penalty for each day (24 hour period) late. If you are submitting your assignment after the due date, it must be emailed to nlerner, lsutliff, and nkuldell AT mit DOT edu. There will be no re-write option on this assignment.
- Begin to familiarize yourself with the content of the second experimental module by reading the front page for the module as well as skimming the introduction to the first day of labwork.
