FAQ

FAQ for Flow Cytometry

Question
  1. How do I arrange to run samples at the Flow Cytometry Core Facility?
  2.  

  3. What are the policies of the Flow Cytometry Core?
  4.  

  5. What fluorochromes/colors can I use?
  6.  

  7. How many cells should I have?
  8.  

  9. How much do the cytometers cost per hour?
  10.  

  11. How long will it take to sort my cells?
  12.  

  13. What should I bring my samples in?
  14.  

  15. Do I have to fix my cells? If so, what should I fix my cells in?
  16.  

  17. What Controls do I need?
  18.  

  19. How do I reduce non-specific binding?
  20.  

  21. What software do I need to analyze my data?

How do I arrange to run samples at the Flow Cytometry Core Facility?

  1. Review the Flow Cytometry Calendar for available times.
  2. Call the flow core at 567-3911 to reserve the desired time slot.
  3. Submit a request through the University Shared Instrument resources website at: https://vpr.uthscsa.edu/coresv2
  4. For EACH experiment requiring sorting or analysis of live cells, a Biosafety Form must be completed and signed by the PI prior to the submission of samples. This can be sent to via e-mail to the core, or be brought into the core before the experiment slotted time.

What are the policies of the Flow Cytometry Core?

What fluorochromes/colors can I use?

  • Each of our instruments has a different configuration and allows for different dyes. For a quick look of the most common fluorochromes, please view the "Instruments and Applications" page. For information of fluorochrome excitation, emissions, wavelengths and spectral overlap, see the BD Spectrum Viewer or the Invitrogen Spectrum Viewer.

How many cells should I have?

  • We advise starting with 10^6 cells for the initial stain for analysis. After staining, we recommend a final volume of 300uL. Fewer cells and larger volumes means longer collection time.

How much do the cytometers cost per hour?

How long will it take to sort my cells?

  • This time will vary depending on how many cells you want to collect. An estimate of the shortest possible sort time can be found in the chart below. This calculation is based from a typical 5000 cells/second aquisition rate.
    Cells to Collect Starting (Source) Percent Positive
    0.1% 1.0% 5.0% 10% 50%
    103 30 min. 0.3 min 0.1 min 0.03 min 0.01 min
    104 33. min 3.3 min 0.7 min 0.3 min 0.1 min
    105 5.6 hr 33.3 min 6.7 min 3.3 min 0.7 min
    106 55.6 hr 5.6 hr 1.1 hr 33 min 6.7 min
    107 555.6 hr 55.6 hr 11.1 hr 5.6 hr 1.1 hr
    108 5555.6 hr 555.6 hr 111.1 hr 55.6 hr 11.1 hr

     

  • If the above times are unreasonable, then you should try to enrich your samples (i.e. magnetic enrichment kits, plastic adherance of unwanted cells)

What should I bring my samples in?

  • Samples should be brought in 12x75mm round bottom polystyrene Falcon tubes (catalog# 352008). NOTE: Same size tubes by other vendors may not be compatible with the O-ring on our instruments.

Do I have to fix my cells? If so, what should I fix my cells in?

  • Potential biohazardous material should always be fixed. Cells should be fixed in a final concentration of 1-2% formaldehyde or 1-2% paraformaldehyde. These fixatives can alter fluorescence properties. An alternative is to fix for 20-30 minutes followed by a wash in PBS. This may help produce more consistent results between experiments.
  • For unfixed cell analysis and cell sorting, special precautions need to be made and the core staff must be notified.
  • Specimens known to be HIV, HCV, HBV, or pathogens higher than than BSL-2 are NOT permitted in the Flow Cytometry Core.

What Controls do I need?

  • The proper controls are needed every time for instrument set up to insure consistent results. There are several types of controls, however they can generally be divided into two main categories.
    1. Experimental Control
      • These consists of positive and "condition free" control. For example, an apoptosis experiment would consisted of an untreated sample and a sample with maximal cell death.
    2. Flow cytometry specific controls
      • Single color control. A sample with only a single color for EACH of your colors is needed for compensation of fluorochrome emission overlap (If you are performing an 8-color analysis, then 8 single-color tubes are needed PLUS one unstained tube)
      • Therefore single color controls must have an adequate signal and percent positive to adjust settings.
      • Single stain controls do not have to be specific for the same marker used in the experiment. (Compensation beads (Spherotech) may be used as an alternative instead of using your precious sample).
      • Negative controls. Negative controls should consist of unstained samples, isotypes, and fluorescence minus one (FMO) fluorochrome staining. Isotypes should be of the same subclass, species and fluorochrome, as the antibodies used in the experiment. The isotypes should also be used at the same concentration and purchased (if possible) from the same manufacturer. FMO staining should include positive staining minus one of the fluorochromes.

How do I reduce non-specific binding?

(This section is from Joanne Lannigan's website from University of Virginia)

 

  • Non specific binding can be due to several reasons.
    1. Too much antibody can increase the amount of non-specific binding of your negative population reducing your signal:noise. If the antibody you are using has not been tittered, than a titration of your antibody should be done to determine the optimal concentration.
    2. Non-specific binding can also be due to Fc mediated binding. You can use IgG of the same species as your antibody to block nonspecific binding. Incubate samples with a final concentration of 2mg/ml of IgG for 5-10 minutes at room temperature before adding specific antibodies. Using monoclonal antibodies specific for Fc Receptors to block Fc mediated binding can also help reduce background binding.
    3. 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 available with which you can easily conjugate your antibody. Molecular Probes (Invitrogen) 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. Other options include conjugation kits available from Prozyme, Inc and the standard do-it-yourself protocols (see http://www.drmr.com/abcon/).
    4. 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 the Molecular Probes "The Handbook" (section 7.6) for a discussion of 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.
    5. Dead cells are notorious for non-specifically binding antibodies. Inclusion of a viability dye (i.e. PI, 7-AAD) 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 can not be fixed when using a viability dye as this will make them permeable to the dye and all the cells will appear dead.

What software do I need to analyze my data?

  • You will need a software package that recognize FCS files (flow cytometry format). The flow core uses FACSDiva (BD Bioscience) and FlowJo (TreeStar) for data analysis. The flow core has a dedicated analysis workstations for each package that users can use free of charge. These two packages are by no means the only packages out there.