Rpmi 1640 medium

Murine 32D cells (DSMZ, Braunschweig, Germany) were cultured in RPMI-1640 medium (PAN Biotec, Aidenbach, Germany) supplemented with 10% fetal calf serum (FCS) and 1% Penicillin-Streptomycin at 37°C with 5% CO₂. Human TF-1 cells (DSMZ, Braunschweig, Germany) were cultured in RPMI-1640 medium supplemented with 20% FCS, 1% Penicillin and 2 ng/ml GM-CSF (Immunotools, Friesoythe, Germany). The transduction of 32D MPL-HA (32D^MPL) cells with retrovirus containing JAK2V617F or CALRdel52 cDNA has been described before (Czech et al., 2019). The transduction of the TF1 cells was performed as follows: First, human TF1 cells were retrovirally transduced with the ecotropic Scl7a1 (Eco) receptor, which made them susceptible for infection with murine retroviruses, and cells were positively selected with neomycin. Retroviruses, which carried the pMSCV-MPL-HA vector, were produced using PlatE cells as previously described (32 (link)). Next, the TF1 Eco cells were transduced with a pMSCV-MPL-HA-generated (TF1^MPL) retrovirus and positively selected with puromycin. Finally, the cells were transduced with retrovirus carrying either the JAK2V617F or CALRdel52 mutation.

MDM were generated as described previously (Pathak et al., 2013 (link)). Briefly, RosetteSep human monocyte enrichment cocktail (Stem Cell Technologies) was used to isolate CD14-positive monocytes from buffy coats (obtained from the blood bank at Karolinska University Hospital, Huddinge) by negative selection. Isolated cells were stained with anti-CD14 antibody (Clone TUK4; DAKO) to determine the purity. Monocytes were allowed to adhere on cell culture plates for 2 h followed by washing of unbound cells. Monocytes were cultured for 6–7 days in RPMI 1640 medium supplemented with 10% FBS and recombinant human macrophage colony-stimulating factor (M-CSF; 50 ng/ml; Immunotools). To assess the differentiation of monocytes into macrophages, changes in cell morphology were monitored by light microscopy (Carl Zeiss, Axiovert 40 C) and cells were stained with anti-CD68 (clone Y1/82A; BD biosciences) and anti-CD14 antibodies. Sample acquisition was performed using LSRFortessa flow cytometer (BD Biosciences).

Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat from healthy donors after informed consent as described previously [27 (link)]. Blood was drawn according to the instruction of our local ethics committee. To receive monocyte-derived DC, cells were separated through two density gradients using ficoll (Lymphoprep-Nycomed, Norway) and OptiPrep Density Gradient Medium (Sigma-Aldrich, München, Germany). Monocyte-derived DC were cultured in RPMI1640 medium supplemented with 10% heat-inactivated, autologous serum and 750 U/ml GM-CSF and 500 U/ml IL-4 (Immunotools, Friesoythe, Germany). The medium was replaced on day + 4 after the DC generation.