Flow Cytometry - DOC by eYhL5doH

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Flow Cytometry or FACS (Fluorescence Activated Cell Sorting)
Introduction to the technique

Flow cytometry is the characterization of single cells as a fluid stream of cells is passed through a
laser beam at high speed (thousands of cells/second). Cells are analyzed and classified on the basis
of both fluorescence and light emission. The analysis is fast and yields information that is
unattainable by other methods. As each cell flows past a focused laser beam (or beams) of
appropriate wavelength(s), light is scattered and detected in both the forward and side (90 degree)
parameters. If the cell contains any molecules that fluoresce from the wavelength of the laser light,
fluorescence is emitted; this allows looking at the cells with the property associated with that
particular fluorescence. This information is used to determine the characteristics of those cells.
Flow cytometry provides data on large numbers of individual cells. The information gathered from
each cell is stored. To allow for multiple different biological or biochemical properties of the cells
to be determined at one time, the cells can be stained with different fluorescent dyes which bind
specifically to the cellular component of interest. Forward scatter gives an indication of a cell's
relative size and side scatter an indication of its texture (granularity). Fluorescence depends on the
binding of the different fluorescent dyes for the property of interest.

Many possibilities, depending on instrumentation, exist for flow cytometric analysis or individual
cell sorting, including, but not limited to:

   * Immunofluorescent measurements for antigen and antibody
   * Determination of cell viability using Dead/Live cell gating
   * Expression of Green Fluorescence Protein (GFP), or ds-Red
   * Detection of apoptosis with TUNEL or Annexin-V
   * Multicolor identification or sorting of bacteria, parasites, neurons, sperm, or RBC’s
   * Quantitative analysis of the number of molecules of a particular antigen found on the surface of
a cell (MESF)
   * Cell division studies using antibodies to bromodeoxyuridine (BrdU)
   * DNA and cell cycle analysis, DNA Index for aneuploidy
   * Mutant isolation
   * Plant cell studies
   * Virus infected cells
   * Sterile sorting into multi-well plates, trays, or onto individual microscope slides with automated
cloning device
   * One, two, three or four way sterile tube sorting

Cell Sorting

Cell sorting involves physically separating (purifying) a specific cell population from the remaining
cells or particles in the suspension for the purpose of performing further experimental procedures on
these purified cells. Sterile sorted cells can be cultured to expand the population. Cells can be re-
stained with other fluorescent dyes to be re-analyzed, processed for RNA studies, or other
experiments to be done to characterize the purified population.

In most cell sorters, cells are passed in a stream of fluid through the flow cell; they pass through a
laser beam and are analyzed in the same way as in standard flow cytometry. The sample stream is
broken into droplets at a fixed break off point. If a cell of interest passes through the laser beam, it


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is identified, and when it reaches the droplet of the break off point, an electric charge (positive or
negative) is applied to the stream. As the droplet leaves the stream it passes through deflection
plates carrying a high voltage and the droplet will be attracted to one of these plates, depending on
the charge it was given. Uncharged droplets pass through to the waste, and deflected droplets are
collected in tubes. In this way one, two, three or four different populations of cells can be sorted
from the one sample. Cells can also be sorted using a cloning attachment, which has the capability
of sorting single cells (or any specified number of cells) into 96 well plates (or any other
size/number of wells), or directly on to a microscope slide.
Sorting Speed

There are a number of factors that determine the length of the cell sort, including cell type, cell size,
cell concentration, sticky or clumpy cells, sort mode (purity or enrichment), and frequency (%) of
the target cells.

Sorting speed is generally around 10,000 - 12,000 cells per second using the 100 micron flow cell
tip for larger or sticky cells, and around 25,000 cells/second for smaller, well-suspended cells using
the 70 micron flow cell tip.

Expected yields are usually 40% of the ideal yield on average. Highly purified cells may be less due
to the number of aborted droplets. Higher yields are possible with less purity or enrichment.

Any BSL 2 material, including tissue from healthy human donors, must be sorted with the aerosol
evacuation unit running, and this adds some time to the sort. The sorting chamber must be
evacuated before removing the sorted cells.

Sample Preparation

As a general requirement, all samples should be prepared in a single-cell suspension form at a
concentration of 6 - 20 million / ml (depending on cell type), after all labelling and filtration steps.
DNA analysis requires fewer cells / ml. Before analysis the samples should be filtered through a 35-
50 micron filter to remove any clumps or aggregates. If this is not performed, and your sample does
contain clumps, it can clog the flow cell. When this happens the clog need to be removed and the
machine re-calibrated.

Appropriate control samples must be included in all experiments. The control could include:

  * A sample of unstained cells
  * Positive and negative controls
  * Compensation (single colour) controls for each stain as cells or compensation beads

There is an excellent link on the UCLA Flow Cytometry web site with procedures for numerous
flow cytometry protocols. That link is: http://cyto.mednet.ucla.edu/.

Fluorescent compensation

For surface staining of cells; usually three controls are needed for each experiment. The controls
must undergo the same treatment (i.e., preparation) as all the tubes in an experiment. An unstained
control is used to detect auto-fluorescence or background staining innate to the cells of interest.
Auto-fluorescence can be a significant problem, particularly in systems that contain
monocytes/macrophages, cultured cells, or activated cells. An isotype control (i.e., where an


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antibody is used that has the same immunoglobulin isotype as the test antibody, but a different
specificity which is known to be irrelevant to the sample being analyzed) is needed to determine
whether fluorescence emitted is due to non-specific binding of the fluorescent antibody. A positive
control is highly desirable (although not always available) to prove that the test antibody and/or
procedure is working properly. The positive control should include cells known to be positive for
the marker of interest.

If more than one colour (fluorescent conjugate) will be used within the same experiment, a single-
colour control must be prepared for each of the dyes used. This will allow for compensation from
spectral overlap between the stains. An unstained control is also important for multi-colour
compensation. In any experiment where two or more fluorochromes (multi-colour analysis) are
used to characterize the cells of interest, the need for fluorescence compensation must be checked.
Fluorescence compensation is the mathematical subtraction of the fluorescence of one fluorochrome
from the fluorescence of another fluorochrome. This is necessary because emission spectra from
multiple fluorochromes can overlap. For this reason, in a sample that contains 2 colours such as
fluorescein, isothiocyanate and phycoerythrin, fluorescence due to the FITC will be detected by the
electronics that are set up to detect fluorescence produced by the phycoerythrin. If this were not
subtracted from the phycoerythrin fluorescence, the phycoerythrin signal would be falsely elevated.
To determine the correct amount of fluorescence compensation, cells stained with only one
fluorochrome-conjugated antibody are analyzed individually. This must be done for each of the
fluorochromes used in the experiment, with an unstained control as the initial region.

To further understand fluorescent compensation, there is a tutorial by Mario Roederer at the
following link: http://www.drmr.com/compensation/index.html



Esimerkkikuvia Marko Pesulta. ppt-slide, tulee erillisenä liitteenä.


Instrumentation in Kauppi Campus
We have four instruments in use currently, two in IMT for routine cell analysis, one in Regea in
clean room environment suitable also for sorting experiments and one in Vactia P3 safety
laboratory.

Beckman Coulter Epics XL-MCL Cell Analysis
(Installed 2003) http://www.beckmancoulter.com/products/instrument/flowcytometry/epicsxl.asp
Located in IMT FM1, 3rd floor, room 3-104




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This instrument will be still maintained at least till the end of July 2010 to enable researchers to
finish their ongoing experiments.

Capability to analyze up to 4 colours of immunofluorescence from a single air cooled laser.
Other multi-colour applications include: multiparametric DNA analysis, platelet studies,
reticulocyte enumeration, cell biology/functional studies and as well as a broad range of research
applications. The instrument is self contained and biohazard safe. The user retains the flexibility to
change filter elements for versatility in research settings.

Accuri C6 Flow Cytometer Cell Analysis (Installed November 2009)
http://www.accuricytometers.com/products/intro/
Located in IMT FM1, 3rd floor, room 3-104




An industry-standard, sheath-focused core facilitates high performance while enabling event rates
of up to 10,000 events/second and sample concentrations over 5x107 cells/ml. Measures the volume
pulled from your sample so that you can calculate cell concentration
Has a fully digital data collection system, allowing complete flexibility to display and analyze data
post-collection, without permanent alteration of your original data file.

Equipped with a blue and a red laser, two scatter detectors, and four fluorescence detectors with
interference filters optimized for the detection of FITC, PE, PerCP-Cy5.5 and APC.
Works with the same reagents you currently use on the market- leading flow cytometers.


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C6 fluidics system is non-pressurized enabling any brand of sample tube that is 12x75 mm or
smaller to be used. This includes microcentrifuge tubes and tubes made of polypropylene or
polystyrene.

CFlow® files can be exported in FCS 3.0 format for seamless importation of your data into most
3rd party flow cytometry software programs including FCS Express and FlowJo.


Liitteeksi pdf-filet:       7820011B CFlow Guide
                            7820023A C6 Start-up & Shutdown Card
                            Accuri C6 Flow Cytometer Instrument Manual Serial #2400 and higher
                            QuickStart Guide Analyze
                            QuickStart Guide Collect
                            QuickStart Guide Statistics

Terms of Usage and Pricing

Please contact kimmo.savinainen@uta.fi if this is your first time using the instruments. Do an
advance reservation for the instrument*.
Usage is allowed only after the first free introduction to the instruments. Further tutorials are
possible with an extra price. All solutions except your sample suspension buffer are included. All
researchers must fill the log book with complete information of the billing address to be used for
charges, from UTA personnel your name and group info (and possible fund number) is sufficient.


User                        Service                                   Fee per hour €
Academic researchers        Instrument usage                          8,00
                            Advanced tutorials                        26,00
Others                      Instrument usage                          16,00
                            Advanced tutorials                        60,00

Table. User fees per hour for Beckman Coulter and Accuri 6 Flow Cytometers. All the bills will be
topped by overheads and VAT when applicable.

*Please be aware that if you cancel your reservation later than one working day before your
reservation, you are still charged for the reserved time. If you need to cancel your reservation,
please do so as early as possible.


BD FACSAria – Cell Analyses and Sorting
Service is available for research purposes at Regea Institute of Regenerative Medicine in FM5.
http://www.regea.fi/services.php?p=8&l=fi
http://www.bdbiosciences.com/instruments/facsaria/index.jsp




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BD FACSAria (BD Biosciences) is a high-speed flow cytometer suitable for the analysis and
classification of cells enabling speeds of up to 70,000 events per second. It is possible
simultaneously to identify as many as 13 fluorescent and 2 chip parameters.

The instrument is equipped with three lasers (Blue 488 nm, Red 633 nm, Violet 407 nm), two
nozzles (diameters 70-- and 100--) and is temperature controlled within the range +4…+42 degrees
centigrade. Software in use is FACDiva Software version 4.1.2.

Cells can be sorted into sample tubes or well-plates or onto microscope slides.

Renting

Prior to the first analysis the research group should go through the test arrangement and complete
the enclosed sample information form. The form is available in PDF or as a DOC file.
The rent for a two-hour run is 107.31 Euros (VAT 0%), in addition to which consumption of fluid
while running and other material costs will be invoiced. The minimal rental is for two hours.

More information is provided by:
Bettina Lindroos, tel. +358 40 190 1789, bettina.lindroos(at)regea.fi
Maria Sundberg, tel. +358 40 190 1794, maria.sundberg(at)regea.fi




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