Icyt sy3200 cell sorter

The cell enrichment and sorting procedures were developed to ensure the appropriate yield of DN cells for our experiment as previously described in Xu et al. (2017) (link). Basically, an aliquot (∼1.5 × 10⁸ cells in 6 mL) of the isolated thymus cells were centrifuged and concentrated to 1× 10⁸ cells/mL in mouse medium and stained with 1 μg/mL PE-conjugated anti-CD4 and PE-conjugated anti-CD8 antibodies for 15 min at RT in dark. 150 μl of PE selection cocktail was then added to the cell suspension, followed by a15 min RT incubation. Seventy five microliter of magnetic nanoparticles was added to the cell suspension and mixed well. After a 10 min RT incubation, cell suspension volume was brought up to 2.5 mL by adding DPBS⁻ containing 2% FBS and 1 mM EDTA. Tubes were then placed into the EasySep™ magnet (STEMCELL Technologies) for 5 min and cells that remained in suspension (∼ 94% pure DN cells) were collected into a new tube. Enriched DN cell suspension and the remaining total thymus cells were stained with 2 μg/mL FITC-conjugated anti-CD8 and APC-conjugated anti-CD4 in mouse medium and DN and DP cell populations were sorted into two 15 mL tubes on an iCyt SY3200 cell sorter (Sony, San Jose, CA) at 15,000 events/s to achieve > 99% purity. By adding the above described enrichment step before cell sorting, the efficiency of obtaining DN cells was significantly improved.