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Frequently Asked Questions

 

I have used the identical equipment somewhere else.
Do I have to be trained again?

Not necessarily. You simply need to have an account set up for you on the equipment that you have been trained to use and be informed of the lab’s specific procedures, e.g., where to store your data files and how often they are deleted from the computer, how to create appointments, etc.

 

What type of samples can be run on the flow cytometers?

Any single cell or particle suspension can be analyzed, which includes mammalian cells, bacteria, yeast, dissociated tissue and polystyrene beads. If your cells or particles are not in single cell suspension, you cannot perform flow cytometry. Cells that are not from whole blood must be filtered. Persons working with adherent cells must make sure that their cells stay in single cell suspension before and during the entire process. In those situations, it is recommended to suspend cells in a product called Accutase, which can be purchased from its creator, Innovative Cell Technologies, or from many of the usual suspects, e.g., Sigma, eBioscience, Chemicon, and probably others. Filtering alone is insufficient to prevent adherent cells from reforming clumps.

 

What should I bring my samples in?

For analysis, samples should be brought in 12 x 75 mm round bottom style polystyrene plastic Falcon test tubes (catalog #352008). Know that the same size tubes from other manufacturers may not necessarily fit the sample O-ring on the analyzer flow cytometers.

 

For sorting, samples should be brought in 12 x 75 mm round bottom style polyproylene or polystyrene plastic Falcon test tubes (catalog #352063 and 352008, respectively) or in 15 ml conical tubes.

 

I need protocols for sample preparation.
Where can I obtain them?

This Web site has a number of links to important and comprehensive protocols. Click on the Protocols & Useful Links section of the facility's Web site to access them.

 

How many cells do I put into each tube?

For analysis, we recommend that you put 1 million cells in 500μl of FACS buffer into each test tube.

 

What controls do I need?

Flow cytometry controls consist of different types and are necessary every time an experiment is performed. Basic flow cytometry controls consist of negative and single color controls. Negative controls consist of cells alone or an isotype control. Single color controls are used for compensation of fluorochrome emission overlap. Single color controls must have sufficient signal to be able to adjust instrument settings. Single color controls do not necessarily have to be the same antibody as the antibody of interest as it is the fluorophore that is being compensated, not the antibody. If the conjugated antibody of interest is not brightly expressed, that antibody would not be an appropriate single color control. Single color controls have been made much easier since some manufacturers sell beads that are designed to bind antibodies, creating the necessary single color controls flow cytometry requires (BD Bioscience Compbead, catalog #55283 for the anti-mouse Ig kappa beads or Bangs Lab Simply Cellular Compensation Standard anti-mouse beads, item #550).

 

Some persons may or should choose to prepare additional controls consisting of fluorescence-minus-one [FMO] controls. FMO controls determine how to analyze the sample, i.e., to assist with gate setting for positive and negative expression and should be considered a must whenever accurate discrimination is essential or when antigen expression is relatively low. Jennifer Wilshire, PdD, gave a great lecture on the importance of FMO controls. FMO controls cannot be used to set compensation.

 

Related to typical flow cytometry controls, positive controls are used to ascertain the activity of an antibody of interest and are required in certain situations. For example, you may want to prove that your cells do not express a particular antigen. In this situation, you should supply a different cell type that expresses the antigen to prove that the antibody is specific to the epitope of interest.

 

Experiments run without the appropriate controls make the interpretation of the information difficult and any resulting conclusions must be considered suspect.

 

Can dye X be run in the facility?

It depends upon the dye's excitation and emission characteristics. The facility is equipped with a variety of lasers though it is not comprehensive. Check the facility's Web site for a summary table of the available lasers. In addition, check out the fluorescent spectrum viewers from Becton Dickinson and Invitrogen.

 

Can dye X and dye Y be run together?

It depends upon the dyes' emission characteristics and the instrument. Again, check out the fluorescent spectrum viewers from Becton Dickinson and Invitrogen.

 

I have potentially infectious (biohazard) specimens. What do I do?

The core facility does not currently accommodate biohazardous specimens. Specimens must be inactivated of pathogens before they are brought into the facility. Commonly, 1-2% paraformaldehyde is used. The laboratory recommends liquid methanol-free EM grade 16% paraformaldehyde from Electron Microscopy Sciences and dilute it using PBS. (10 x 10ml glass ampules are $22.50). Users should be aware that Invitrogen has a variety of LIVE/DEAD Fixable Kits that use an amine reactive dye that allows users to fix cells and to subsequently discriminate live from dead cells in the original sample. Other than preserving cells, these kits have the added benefit in that they inactivate pathogens.

 

I would like to sort cells but I am uncertain how to do that.
What do I do?

Persons that have never sorted cells before must first contact William King, the lab manager. He will ascertain the feasibility of your sort goal, provide you with guidelines associated with sorting cells and create an appointment for sorting with you.

 

How can I reduce nonspecific binding?

This section is taken pretty much intact from Joanne Lannigan's Web site at the University of Virginia — thanks, JoJo!

Nonspecific binding can be due to several reasons.

 

Too much antibody will increase the amount of nonspecific binding of your negative population, which will reduce the signal-to-noise ratio. You should perform titrations for every antibody you use to determine the optimum concentration.

Nonspecific binding can also be due to Fc-receptor binding. Use IgG of the same species as your antibody of interest to block nonspecific binding. Incubate samples with a final concentration of 2 mg/ml of IgG for ~10 minutes at room temperature before adding antibodies. Using monoclonal antibodies specific for Fc receptors to block Fc-mediated binding can also help reduce background binding.

 

The use of directly conjugated antibodies can also reduce the amount of non-specific binding. If your antibody is not commercially available as a directly conjugated antibody, there are a number of simple procedures with which you can easily conjugate your antibody:

 

1. Invitrogen/Molecular Probes offer conjugation kits for their Alexa dyes which are very simple to perform. In addition, they also offer the Zenon labeling kits, which utilize fluorochrome conjugated Fab' antibodies directed against the specific isotype of your antibody. A simple 10-minute incubation is all that is required.

2. Other options include conjugation kits available from Prozyme, Inc. and the standard do-it-yourself protocols.

 

3. The use of a biotinylated antibody with a streptavidin fluorochrome conjugate can be a source of nonspecific binding in some cells. Biotin is a component of normal cellular metabolism, and as such, there will be truckloads of it within mammalian cells. In addition, see this e-source for a discussion of the nonspecific binding properties of streptavidin associated with its Arg-Tyr-Asp (RYD) tripeptide sequence which mimics the Arg-Gly-Asp (RGD) binding sequence of fibronectin.

 

Dead cells are notorious for non-specifically binding antibodies. Inclusion of a viability dye such as DAPI, PI, 7-AAD, or TO-PRO-3 iodide, among others, in your assay will allow you to exclude the dead cell population from your analysis. You must make sure that your viability dye is compatible with the other fluorochromes in your sample. Also, cells cannot be fixed when using a viability dye as this will make them permeable to the dye and all the cells will appear dead. As mentioned previously, users should be aware that Invitrogen has a variety of LIVE/DEAD Fixable Kits that use an amine reactive dye that allows users to fix cells and to subsequently discriminate live from dead cells in the original sample. Pathogens are inactivated in the process.

 

How often are data files removed from the computers?

No data files are allowed to be stored on the computers in the lab. After running your samples you should immediately transfer your data files onto some other media source, i.e, a thumb drive or transfer them to the network and subsequently transfer to your own computer. Your data and your experiments are ultimately your own responsibility.

 

How to schedule instrument use:

Scheduling for all equipment and services in the core facility is accomplished using a web-based electronic calendar.

 

How much do the cytometers cost per hour?

A current schedule of the facility's fees can be viewed on this Web site. Users will be billed for the time that they reserve or the time that's actually used, whichever is greater.

 

William King is soley responsible for the content of this site. Comments, concerns and questions regarding it should

be addressed to him.

 

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