5.2. DirectCell™ Protocol

For a small number of cells, such as immune cell fractions (e.g., 5,000-50,000 cells), and tissue samples (less than ~1 mg), we recommend FACS sorting or magnetic-bead isolation without any DNA purification step. Using sorted cells directly increases the sensitivity of CDR3 clonotype detection as compared to DNA isolated from small biological samples. Refer to the guidelines in this section for preparation of these samples.

To use small samples directly in the assay, follow the procedure below:

1. Purify immune cells (e.g., from blood or dissociated tissue samples) and store the cells at -80C in small volume (5-10 ul) of PBS or Tris buffer without EDTA. FACS sorting may require spinning down the cells and removing excess supernatant over 10 ul.

2. Follow the standard AIR protocol for primer extension and follow-up step using cell suspension instead of DNA.