Leukemic Subpopulation Harvest: A Method for Spatial Separation of Leukemic Cell Subpopulations from 2D Co-culture

1. Separating 3 Subpopulations within Co-culture

1. Collection of suspension (S) tumor subpopulation.
   1. Aspirate media from co-culture plate with pipette and gently re-apply the same media to rinse the plate and collect media containing leukemic cells in a 15 ml conical tube. The leukemic cells collected are the S subpopulation.
2. Collection of Phase Bright (PB) tumor subpopulation.
   1. Add 10 ml fresh media back onto co-culture plate. Rinse vigorously by pipetting added media up and down approximately 5 times to remove adherent leukemic cells but not hard enough to dislodge adherent BMSC/OB component.
   2. Aspirate with pipette and collect media in a 15 ml conical tube. The collected cells are the PB subpopulation.
3. Collection of Phase Dim (PD) tumor subpopulation.
   1. Rinse plate with 1 ml PBS to remove remaining media. Trypsinize co-culture plate with 3 ml trypsin and place into 37 °C incubator for 5 min.
   2. Remove plate out of incubator and gently tap sides of the plate to dislodge adherent BMSC/OB.
   3. Add 1 ml fetal bovine serum (FBS) and pipette up and down 3-5 times to break apart large cell aggregates.
   4. Collect media with cells in a 15 ml conical tube. These cells are the unpurified PD subpopulation with BMSC/OB as well.
4. Centrifuge 3 isolated subpopulations at 400 x g for 7 min. Aspirate and discard supernatant then individually resuspend pellets in 1 ml pre-warmed media. Cells are ready to be loaded onto a G10 column.