Easy Sep for Hassle-Free Cell Separation

Protocol Overview

We grew HT29 cells, trypsinized them and followed the kit instructions using live cells. Briefly, we resuspended the cells into a 2 x 10^6 cell/ml suspension in a 5ml round bottomed tube (not supplied in the kit). Our primary antibody was not FITC-conjugated, so we separated the antibody incubation steps into 2 parts: First, the primary anti-SSEA5 mouse monoclonal at 1ug/ml (Genetex) and then a goat anti-mouse FITC secondary antibody at 1ug/ml (Sigma). We then added 100ul of Selection Cocktail from the kit and incubated at room temperature for 15 minutes. We then added 50ul of the magnetic beads and let the tube sit for 15 minutes. We added buffer to bring the volume up to 2.5 ml and placed the tube into the Easy Sep Magnet (not part of the kit). We then poured off and collected the supernatant while the tube was still in the magnet. We carried out two additional washes of the tube with 5 minute incubations in the magnet, always collecting the supernatant for SSEA5- cells. At the end, the tube in the magnet had SSEA5+ enriched cells. We were able to photograph the two populations in the attached image.