I have used the identical equipment somewhere else. Do I have to be trained again?
Not necessarily. We simply have to set up accounts for you on the equipment that you have been trained to use and inform you of our specific procedures, i.e., 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 include 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, we recommend suspending 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 bench top analyzers.
For sorting, samples should be brought in 12 x 75 mm round bottom style polyproylene plastic Falcon test tubes (catalog #352063).
I need protocols for sample preparation. Where can I obtain them?
The facility's web site has a number of links to important and comprehensive protocols to include 'Protocols in Cytometry'. Click on the 'Protocols and 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 500 μl 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, which facilitate 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 are used to identify 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. Go to http://www.urmc.rochester.edu/wnyfug/pdf/wilshire-2004.pdf#search=%22FMO%20controls%22 for further explanation 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 wide variety of lasers. Check the facility's web site under the 'Flow Cytometers' section for a summary table of the facility's 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. Check out the fluorescent spectrum viewers from Becton Dickinson and Invitrogen.
I have potentially infectious (biohazard) specimens. Can I sort or analyze them?
In many but not all cases, the answer is "yes". The information below summarizes our current policies. If you have questions, please contact the facility personnel directly.
For high speed sorting and analyzing using the MoFlo or FACSAria sorters, we have established protocols that enable the sorting of most well characterized human cell lines, and some types of primary human cell suspensions.
Currently, we CANNOT sort any samples that are known or strongly suspected to be infected with HIV, HCV, HBV or other pathogens requiring higher than standard BSL-2 level containment or other special precautions.
We CAN and will accommodate requests for sorting and analyzing of many low to intermediate virulence pathogens, including Toxoplasma, Cryptococcus, selected Gram positive organisms (including most Staphylococci, Streptococci and Listeria spp.), selected Gram negative organisms (E. coli, most Salmonella and Yersinia spp.), and some attenuated mycobacteria (BCG, M. smegmatis, double auxotrophic strains of M. tuberculosis that have been reclassified to BSL-2). Other organisms in addition to the ones mentioned may also be considered, pending review of specific requests.
We CAN sort most established human cell lines, provided that these are not known or strongly suspected to harbor potentially hazardous transmissible agents such as infectious virions of clinically relevant viruses such as HPV, HIV or hepatitis viruses. In general, cell lines obtained from ATCC are acceptable for sorting, as are many other extensively passaged human cell lines obtained from other sources. However, please note that all requests for sorting of human cell lines require that you first fill out a brief application form and submit it to the FACS Facility staff for review (see below).
Primary human cells and tissues that can be sorted are currently limited to cell suspensions from blood bank derived specimens that have been pre-screened and found to be negative for infectious blood borne viruses (e.g., HIV, HCV and HBV). Other primary human cells CANNOT be sorted by FACS in our facility under our current protocols. We offer a variety of magnetic sorting options that may provide a suitable alternative, and other options may be available on campus for those who require high speed single cell sorting of primary human cell suspensions. Please contact the facility personnel for further information on available options.
All requests for sorting of human cells or potentially hazardous pathogens require that you submit a completed biohazard questionnaire form. The form can be downloaded from our Website (http://www.einstein.yu.edu/FACS/) by clicking on the "Forms" link on the menu list of the Facility's home page. The completed form, which should not take more than about 5 minutes to fill out electronically, must be submitted at least one week in advance of any scheduled biohazard sort so that the request can be reviewed. If there are any potential problems with a request that preclude immediate approval, we will notify the investigators and work with them to resolve the issues if possible.
Fees for biohazard sorting will include a 20% surcharge in addition to the rate for standard cell sorting procedures (as currently posted on the Facility’s website).
For Analyzing with Analyzers, , the core facility does not currently accommodate biohazardous specimens for analyzers. Any pathogens contained within specimens for analysis must be inactivated before they are brought into the facility. Commonly, 1-2% paraformaldehyde is used for this. We recommend purchasing liquid methanol-free EM grade 16% paraformaldehyde from Electron Microscopy Sciences and dilute it using PBS. (10 x 10ml glass ampules are $22.50). (http://www.emsdiasum.com/microscopy/products/chemicals/paraformaldehyde.aspx). Users should be aware that Invitrogen has a variety of LIVE/DEAD Fixable Kits that use an amine reactive dye that allow 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. Flow cytometers housed in Einstein's Center for AIDS Research (CFAR) can accommodate unfixed specimens containing HIV and a variety of other biohazardous pathogens. Contact Sujatha (Suja) Nagaraja (CFAR FACS Technician, ext. 3226) for more information.
Biohazard Sorting and Analyzing Time and Locations:
Wednesdays, Price Center 159B (FACSAria)
Thursdays, Chanin 308 (MoFLo XDP and/or FACSAria)
I have never sorted before but I cannot create an appointment on the calendar. Why?
Persons that have never sorted cells before must first contact one of the facility's staff first. We will ascertain the feasibility of your sort goal, provide you with comprehensive guidelines associated with sorting cells at Einstein and allow you the permission to create a sort appointment in the scheduling software.
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 (www.probes.com) offers conjugation kits for their Alexa dyes which are very simple to perform. In addition, they also offer the Zenon labeling kits, which utilizes 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 (see http://www.drmr.com/abcon/).
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 http://www.probes.com/handbook/sections/0706.html 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, 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 allow 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?
First of all, even though we will be maintaining a backup of all data generated on the facility instruments, we request that you transfer your data to your own storage media right after each experiment that you do. We cannot guarantee that your data will be safe or available to you when you want them if you fail to do this.
In order to keep the Facility's instruments running properly, we need to remove data files from the computer hard drives on a regular basis. For this reason, all data will be backed up from instrument hard drives at the end of each month (generally between the 26th and the last day of each month), and then immediately deleted. The data will be stored on our backup station (which has multiple hard disks and a RAID system to prevent data loss) in the Facility for three years, after which it will be permanently deleted without further notification. Access to the archived data will be only through the facility staff. There will be a charge for data retrieval (currently set at $50/hour). Users requesting data file retrieval must provide the precise date(s) on which the files were generated, and the identity of the machine used to generate the files. Also, users requesting this service must provide the facility with appropriate storage media on which to copy the files (e.g., USB flash drive for smaller data sets, or external hard drive device for large data sets; alternatively, for data sets smaller than ~2 GB we can copy the files to Reststop for you).
How to schedule instrument use:
Scheduling for all equipment and services in the core facility is accomplished using an online calendar which can be found by clicking on the 'Scheduling' link on the facility's web site. General rules are as follows:
♦ Scheduling is on a first-come, first-served basis.
♦ For sorting, appointments less than 60 hours in advance of the appointment cannot be created or otherwise modified by users themselves. In that situation, if you see sort time that you would like to utilize or you would like to modify an existing appointment, contact the operator and they will create or modify the appointment for you.
How much do the cytometers cost per hour?
A current schedule of the facility's fees can be viewed on the facility's web site. Click on the 'Facility Rates' link in the left-hand column. Users will be billed for the time that they reserve or the time that's actually used, whichever is greater. There is a minimum 1-hour charge for use of any of the equipment or services in the facility.