GSM6336368: Mac4_DMSO_d14; Mus musculus; RNA-Seq
For macrophage isolation, F4/80+ cells were obtained by using the Anti-F4/80 MicroBeads UltraPure kit (Milteny, 130-110-443) following the manufacturer's instructions. Approximately 2x107 single-cells from the tumour were stained with a mix of 180μl MACS buffer and 20 μl of Anti-F4/80 MicroBeads for 15 minutes at 4°C in the dark. During this incubation, an LS column was placed onto a MidiMACS separator where magnetic field was applied, and the column was rinsed with 3ml of MACS buffer. Cells were washed twice with 4ml of MACS buffer and resuspended in 1ml prior to proceeding to magnetic separation by applying the cell suspension onto the column. Columns were washed thrice with 3ml of MACS buffer before being carefully removed from the separator and placed on a FACS tube. 4.5ml of MACS buffer was added onto the LS column and the plunger was firmly pushed to elute the F4/80+ cell fraction which had been retained within the column due to the F4/80+ positive selection. To increase the purity of the cells, the eluted fraction was applied to a second round of separation with a new LS column.