For situations where sample size is limited and there is a need to isolate multiple cell types, one may wish to perform a sequential separation procedure. This avoids dividing the sample and ensures higher recovery of purified cell populations. Sequential separation is ideal for chimerism analysis, which is typically performed on small blood samples and requires techniques that can isolate more than one cell type from a single starting sample for lineage-specific analysis.

Most of our sequential separation protocols can isolate 3 cells types from 1 sample, as follows:

1. The entire sample is labeled with an antibody targeting the first cell type, these cells are then coupled to magnetic nanoparticles and the sample is placed in the magnet. The supernatant with unlabeled cells is removed to a new tube, leaving the first desired cell type in the magnet.
2. The unlabeled fraction from the first separation can then be labeled with an antibody targeting the second cell type. These labeled cells are isolated as described above, and the supernatant is again removed to a new tube for the final separation step.
3. The third cell type is isolated in the same manner, and the supernatant can simply be poured off.

This method can be applied to virtually any combination of cell types desired. At STEMCELL Technologies, we have focused on optimizing various combinations specifically for HLA labs, but the technique can be used in many applications.

General Tips for Sequential Separation:

• Positive selection of a desired cell type must always be performed on an unlabelled cell population; either (a) the original starting sample, (b) the poured-off fraction from a positive selection, or (c) the poured-off fraction or enriched cell population from a negative selection.
• We generally recommend isolating the most rare cell type first (to minimize cell losses), and the most frequent cell type last (however this convention cannot always be followed, please contact us for recommendations).

Advantages of Sequential Separation:

• Allows you to work from a smaller starting volume
• Maximizes cell recovery
• Quick and easy way to isolate multiple cell types
• Can be automated using RoboSep to avoid cross contamination

For a poster detailing a sequential separation procedure to isolate B cells, T cells and myeloid cells for chimerism analysis, please see here.

Please see our website for more information on EasySep™ and RoboSep™.