Anti human cd33 microbeads

On day 6–7 of PBMC culture, suspension and adherent cells were removed from T25 flasks (Detachin, San Diego, CA, USA; Genlantis and gentle cell scraping), collected by centrifugation and incubated with anti-human CD33 microbeads (Miltenyi Biotec, Sydney, NSW, Australia) for 15 min at 4 °C before being washed and resuspended in 500 μl magnetic-activated cell sorting buffer before application to magnetic-activated cell sorting LS Columns (Miltenyi Biotec). Cells were washed three times in magnetic-activated cell sorting buffer. To collect the attached CD33⁺ cells, the columns were removed from the magnet and washed twice with 5 ml magnetic-activated cell sorting buffer; an initial collection via gravity flow and a final wash expelled using the plunger.

Induced M-MDSCs (iM-MDSCs) were generated from CLL autologous myeloid precursors with a protocol modified from Lechner et al. [21 (link)]. After enriching CD33⁺ cells from PBMCs using antihuman CD33 MicroBeads (Miltenyi Biotec), cells (1 × 10⁵ cells/ml) were suspended in complete culture medium supplemented with IL-6 (10 ng/ml; R&D Systems), IL-10 (10 ng/ml; R&D Systems) and GM-CSF (10 ng/ml; R&D Systems), and then cultured for 7 days in a Nunc UpCell 12 MultiDish (Thermo Scientific). In some instances, TNFα (20 ng/ml; R&D Systems) was added at the initiation of culture. After the incubation period, adherent cells were washed and collected from Nunc UpCell 12 MultiDish by cooling plates to below 30 °C, thereby making the culture dish surfaces hydrophobic and detaching adherent cells without altering surface molecule expression [22 (link)].