Protocols

Basic Sorting Buffer

  • 1X PBS or RPMI
  • 1mM EDTA
  • 25mM HEPES pH 7.0
  • 0.5-2% BSA or FBS

Importantly the presence of dead cells and/or debris in the sample greatly reduces almost all the aspects of the sort, including efficiency, purity, accuracy and yield. This is in part due to the fact that 1) dead cells have higher auto florescence compared to live/healthy cells and will obstruct the detection of dim fluorescing populations; 2) dead cells are more subjective to non-specific binding of antibodies and dyes resulting in inaccurate selection and sorting of false positives, which affects the purity of the sort; and 3) the presence of free DNA, released from dead cells in the sample leads to cell clumps and clogs, which can interrupt an jeopardize the entire sort. Also, data from clumped cells is aborted and the events are discarded by the instrument. A higher percentage of cells in clumps reduces the final cell count.

Samples with large amounts of dead cells and/or debris must be cleaned before use. Depending on the specific problem, this can be done by either filtering the sample prior to sorting and/or adding DNAse to the sorting buffer. If the sample cannot be cleaned, it might help to do a viability stain using Propidium Iodide to discriminate live from dead cells and subtract the dead cells from the population. While the addition of Propidium Iodide will help resolve the populations, it is important to realize that it adds an additional parameter, which must be taken into account when selecting fluorochromes.