5.2. DirectCell™ Protocol

For a small number of cells, such as immune cell fractions (e.g., 500-50,000 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. Collect cells in 1xPBS or any commonly used buffer without magnesium or EDTA. Transfer the cells into 1.5-ml or 0.5-ml test tubes, spin down at low speed (e.g., 500g for 5 min), and remove excess supernatant from the cell pellet, keeping the residual volume in the test tube 10 µl (to prevent cell loss due to complete removal of supernatant).
   OR
   Prepare dissociated tissue samples (e.g., by collagenase treatment). Adjust the volume of each sample to 10 µl. Depending on your cell collection method, this may require spinning down the cells and removing excess supernatant over 10 µl.

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