The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Investigator Responsibilities

  1. Cells used to set up the instrument must be the same or very similar to the experimental samples. For example, you cannot use resting lymphocytes to set up the instrument and then run a cell line for analysis. The autofluorescent properties of the cells types are too dissimilar. Setting up the instrument consists of running a negative control and compensation tubes so that amplifier gains can be properly set for subsequent samples.
  2. The least autofluorescent cell type should be used to setup the instrument when running lymphocytes from different tissues. Autofluorescence is generally lowest in thymocytes and gets progressively higher in lymph node and spleen lymphocytes. If spleen cells are used to setup an experiment for thymocytes, the thymocytes may appear to be squashed against the baseline of the fluorescent histograms.
  3. The cell prep must be as clean as possible regarding debris and aggregates. Large amounts of debris and/or red blood cells can obscure the lymphocyte population making forward scatter and side scatter amplifier gain settings difficult to set correctly. The operator will attempt to use the same settings for each run of the same type of experiment. This does not guarantee that the data will fall exactly in the same place and in some instances requires small changes in amplifier settings in order to place the population of interest in the bitmap of FSC versus SSC.
  4. If cells have aggregated to the point that you can see them in the sample tube, the sample will plug the flow cell on the instrument. In most instances, the operator can dislodge the plug and the instrument performs as it did before the plug. Sometimes a partial or complete plug remains. If that happens, the flow cell must be dismantled for cleaning and the instrument must be realigned with the laser. The settings used for the previous samples may not be valid, forcing the operator to rerun the setup tubes and possibly all of the previous tubes. If the flow cell must be dismantled during a sort, the investigator will lose an hour of sort time while the instrument is setup again for the sort. It is important to filter aggregated samples with nylon mesh before running them on the instrument.
  5. The investigator is responsible for having the correct cell density (1 X 106 per ml) and volume (minimum of 300 ul) per tube for analysis. Densities and volumes less then the recommended ones cause difficulty in setting up the instrument. Cell density for sorting should be between 5 X 106 and 20 X 106 per ml. See suggestions for running samples for more information.
  6. The investigator is responsible for bringing the correct compensation tubes for running multi-color experiments. See Compensation Controls for Flow Cytometry for more information.
  7. The person accompanying the tubes should have intimate knowledge of the samples and what the data may look like when correctly run.
  8. The person accompanying the tubes is responsible for making sure that the data look correct on the instrument display screen once the operator has made the final adjustments to amplifier gains and compensation settings. If the instrument needs adjustment, the operator should be informed so that changes can be made to bring the settings into compliance with the investigator's parameters.