ABER FLOW CYTOMETRY

GUIDELINES FOR SAMPLE PREPARATION

  1. The aims of the experiment must be clearly defined and explained before any samples are analysed.
  2. The sample must be safe. In all of our flow cytometers an aerosol of the sample may be produced. It is therefore recommended that any hazardous samples (pathogenic microorganisms / blood samples etc) are fixed (e.g. in 70% ethanol) or rendered safe in some other approved way, prior to analysis. It is ESSENTIAL that the operator is made aware of the nature of the sample (i.e. the organism(s) involved, the origin of the sample, the stains used, hazardous chemicals in the buffer / growth media etc.).
  3. The approximate particle concentration must be known. Ideally the sample concentration should be between 105 and 107 particles.ml-1. If the sample concentration is thought to be close to or known to be above 107 ml-1 provide a suitable (filtered) diluent. (Lower concentrations can be analysed but the analysis time will be long and the signal to noise ratio may be unfavourable).
  4. Ideally, the particles of interest should represent more than 1% (and preferably more than 10%) of the total particles present. (If the percentage of particles of interest is lower than this analysis times will be long and/or the part of the histogram of interest will be defined by only a few 100 or 1000 events).
  5. The approximate particle size must be known. (This is important in order to have some idea of settings to use as a starting point; unstained particles smaller than 0.5 um will be very difficult or impossible to analyse.)
  6. The size of the largest particles should be less than 50 um. (If your sample could contain any particles larger than 50 um it is essential that it is filtered through a 50 um or smaller mesh prior to analysis to avoid time-consuming and potentially costly blockages. This is particularly important when environmental e.g. soil samples are used or when the cells have been prepared from plant or animal tissues. It is not usually a problem with microbial samples, however a few seconds of microscope work could save a lot of time and money.
  7. The optimal excitation and emission wavelengths of any stain(s) used must be known. If the emission is in the visible region of the spectrum you should check that the particles (not the suspending medium) are fluorescent using a fluorescence microscope.
IF THE ABOVE GUIDELINES PRESENT A PROBLEM PLEASE ASK FOR ADVICE.
HAZEL DAVEY

FCM homepageFlow Cytometry Home Page