Comparative analysis of macrophage feeder systems reveals distinct behaviors and key transcriptional shifts in chronic lymphocytic leukemia cells via coculture
Comparative analysis of macrophage feeder systems reveals distinct behaviors and key transcriptional shifts in chronic lymphocytic leukemia cells via coculture
Cocultures of CLL patient cells (n = 3) and primary human macrophages, THP-1 macrophages, NLCs, and mBMDM were set up as described in the manuscript. After 5 days of coculture, the medium of cocultures was collected, and containing CLL cells were purified using CD19 MicroBeads (Miltenyi Biotec), which magnetically label CD19+ cells and separate them via the magnetic field of a MACS Separator. The purification was performed using MACS LS Columns.  Primary and immortalized macrophage cells were used: mBMDM, NLC, primary human macrophages (HD-MDM), and THP-1 macrophages.  NLCs were generated by seeding patient-derived PBMCs and adherent cells were harvested after 14 days.  THP-1 monocytes were differentiated into macrophages via 100 ng/ml PMA for 48 h.   mBMDM were harvested from femurs and tibias of 8- to 12-week-old mice. Bones were flushed and cells were passed through a 100 μm sterile cell strainer. Erythrocytes were lysed with  ACK buffer and resulting cells cultured in DMEM (+ 1 % P/S, 10 % FBS) containing 20 ng/ml murine M-CSF  to pre-differentiate bone marrow stem cells into adherent M0 murine macrophages. After a minimum of 7 days, mBMDM were harvested.  HD-MDM were generated from buffy coats of healthy donors. After PBMCs isolation cells  were seeded in differentiation medium consisting of RPMI containing 10 % FBS, 1 % P/S, 50 ng/ml M CSF, 1 % NEAA, 1 % Na Pyruvate, 2 mM. Glutamax.  After 7 days, cells were collected.  For the isolation of RNA for Next Generation mRNA sequencing, the RNeasy Plus Mini Kit (Qiagen) was used according to the manufacturer's instructions. Samples were processed and sequenced PE100, 35M reads per sample at the Cologne Center for Genomics according to in-house standards