FACS Facility - Frequently asked questions
How should cells prepared for analysis or sorting?
Cells should be suspended in an appropriate medium. The concentration may vary, but 105 - 2 x 107 per ml is recommended, lower concentrations result in longer time for analysis and sorting. (Sorting processes 10-60µl of your sample per minute, so you can easily calculate how long it takes to have the amount of cells you want.)
However, higher concentrations increase the risk that the cells clump together which is bad for analysis and still worse for sorting. (In any case, you have to filter your sample before sorting.) Some types of cells tend more to clump than others. If this is a problem, try to reduce it, for example by adding more serum to the suspension medium.
Which controls shall I prepare for particle analysis?
It is wise also to prepare a separate sample with negatives only (unstained particles, unlabelled cells), so that we can easily discriminate between positives and negatives. In case of multi staining experiments, provide us with also with single stained samples for every type of stain. This is especially important for your first experiment and may be omitted when we have sufficient experience with your type of probes.
At which temperature are the cells sorted?
Regularly, we sort at room temperature. This keeps cells viable but metabolism is low, so that the cells don´t become anoxic during the procedure. Cooling down near zero degree centigrade will shock cells, so that they will need more time for recultivation afterwards. We do thought have the instrumentation for keeping the cells cool, if this it would be necessary for your experiment.
Are the cells sterile after sorting?
We do not call them sterile, but aseptic, which means that the risk of contamination is low. Actually, we have had no problems when recultivating cells after sorting, but the use of antibiotics is most desirable.
Beside my sample, what shall I prepare for a sorting experiment?
You need control samples as mentioned above (“Which controls shall I prepare …”) and something where you want the cells to be sorted in, such as medium. (There should be some medium already in the tubes where the cells will be collected, as no cell likes to stay in plain buffer for a longer period).
How diluted are the cells after Sorting?
With the 70µm nozzle, each cell is in a volume of roughly 1 nl (nanoliter), this will make a final concentration of about one million cells per ml plus the volume of your medium you had put in the tube before.
From the 100µm nozzle, you will get a threefold higher dilution.
What can we do if I need a large amount of sorted cells?
Actually, the sorting procedure is pretty fast, but problems may arise if the fraction of your desired cells is very low compared to the total. For long-time sorting, you should split your samples, so that the cells need not to stay at unfavourable conditions for many hours. Alternatively, you may work with a very concentrated sample and play with the instrument´s thresholds so that the machine will only see the cells you want and can sort much faster. But then, unwanted cells will contaminate your sample, so that you have to sort a second time for purity. (This means for example two times sorting of one hour each, instead of one sort of ten hours.)
What does CV mean?
Even signals from identical particles will distribute to some extent around their mean. The CV (coefficient of variation) gives the standard deviation (s) of the distribution relative to the mean (m) of the population. CV=(s*100)/m
What does „Stain Index“ mean?
It is an index for the possibility to distinguish a positive signal from background. It compares the distance between the means of positives and negatives with the spread of the negatives