Generation of primary cells

4.1.9.1. Generation of dendritic cells from bone marrow

Bone marrow cells are pluripotent cells, from which each hämatopoetic cell can be generated. Mice were sacrificed by cervical dislocation and femurs were prepared by flushing with a syringe with a 24 G cannula until the bone was completely white. Marrow clumps were disintegrated by pipetting up and down with a 5 ml serological pipette. The resulting suspension was transferred to 50 ml tubes. To separate cells from other bone marrow components, suspensions were spun down at 1,300 rpm for 6 min at room temperature. Red blood cell contaminations were removed with 5 ml blood cell lysis buffer. After an incubation time of 5 min, the reaction was stopped with 10 ml RPMI complete medium followed by centrifugation for 6 min at room temperature and 1,300 rpm. Then the marrow cells were resuspended in 5 ml RPMI complete medium and the cell number was determined in a Neubauer hemocytometer (4.1.6).

Cultivation and differentiation of bone marrow derived Flt3 cultures

The bone marrow cells were initially seeded at a density of 1.5 x 10⁶/ml in RPMI complete medium in a cell culture bottle. Under the influence of a Flt3 ligand (FMS-like tyrosine kinase 3 ligand) with a concentration of 35 ng/ml, they differentiated into pDCs and cDCs.

The cultures were maintained under an atmosphere of 37°C, 100% humidity and 7.5%

CO₂. Cells were harvested on day 7 and seeded at a density of 2 x 10⁶/ml (4.1.6) for experiments. The phenotype of the generated dendritic cells was analyzed by FACS (4.7).

These cultures typically contained 30-50% CD45RA+CD11c+ pDCs, 30-50% CD45RA-CD11c+ cDCs and 15% CD11c-cells.

Cultivation and differentiation of bone marrow derived macrophages

Cells were seeded at a density of 0.5 * 10⁶/ml in RPMI complete medium in a 10 cm petri dish. Under the influence of a M-CSF ligand (Macrophage Colony Stimulating Factor) with a final end concentration of 20 ng/ml, they differentiated into macrophages. The cultures were maintained under an atmosphere of 37°C, 100% humidity and 7.5% CO₂. After three days, 20 ng/ml M-CSF was additionally added. After 5 days, the non-adherent cells were first collected and then incubated for 10 min at 37°C with 5 ml PBS^def/3% FCS/ 2 mM EDTA to detach the adherent cells. Cells were seeded at a density of 1 x 10⁶/ml (4.1.6) for experiments. The purity of the cells was analyzed by FACS analysis (4.7).

Cultivation and differentiation of bone marrow derived GM-CSF cultures

Cells were seeded at a density of 0.3 * 10⁶/ml with RPMI complete medium in a cell culture bottle. Under the influence of a GM-CSF ligand (Granulocyte macrophage colony-stimulating factor) with a final end concentration of 10 ng/ml, they differentiated into cDCs.

After three days, 5 ng/ml GM-CSF was additionally added. The cultures were maintained under an atmosphere of 37°C, 100% humidity and 7.5% CO2. Cells were harvested on day 7 and seeded at a density of 2 x 10⁶/ml (4.1.6) for experiments. These cultures typically contained more than 90% cDCs (CD11c+, CD11b+, B220-) (4.1.6).

4.1.9.2. Isolation of PBMCs

PBMC (peripheral blood mononuclear cells) (from the Department for transfusion medicine from the clinical centre at the Philipps University Marburg) were generated from heparinized blood of healthy donors by density centrifugation over the carbohydrate polymer Ficoll (LSM 1077, PAA). This yielded a population of mononuclear cells at the interface that was depleted of red blood cells and most polymorphonuclear leukocytes or granulocytes. The resulting PBMC population consisted mainly of lymphocytes and monocytes. The cells were layered over Ficoll and centrifuged over 30 min at 2,000 rpm without brake. Red blood cells and polymorphonuclear leukocytes or granulocytes are denser and were thus centrifuged through the Ficoll-Hypaque TM, while mononuclear cells consisting of lymphocytes together with some monocytes banded over it and could be recovered at the interface.

PBMCs were harvested, washed two times with PBS^def and resuspended in 20 ml RPMI complete medium without FCS. Finally, cell numbers were determined in a Neubauer

hemocytometer (4.1.6), and cells were seeded with 4% human AB Serum (after stimulation the final end concentration is 2%) onto a 96 well plate at a density of 3 x 10⁶/well. Cells were maintained at 37°C in a 5% humidified CO₂ environment.

4.1.9.3. Isolation of monocytes from human blood

Human monocytes were isolated from buffy coats of healthy blood donors (Pauligk et al.

2004). The term ‘centrifugal elutriation’ describes a technique which involves the balance between a centrifugal force generated by the spinning rotor and a centripetal flow of liquid within a separation chamber. Cells present in the separation chamber are found in a position at which the two forces acting on them are at equilibrium. The position of each cell is determined by its size, shape and density. Because the chamber's geometry produces a gradient of flow rates from one end to the other, cells with a wide range of different sedimentation rates can be held in suspension. By increasing the flow rate of the elutriating fluid in steps, or by increasing the rotor speed, successive populations of relatively homogeneous cell sizes are washed from the chamber. Each population contains cells which are larger or more dense (i.e., faster sedimenting) than those of the previous fraction (Figdor et al. 1983). The following correlation exists between the flow rate and the rotor speed (Beckman, Instruction Manual):

F = X x D² x (RPM x 10^-3)^-2

F = flow rate at the pump in ml/min X = 0, 0511 (Standard chamber) D = cell diameter in µm

RPM = rotor speed pro minute

By using a constant rotor speed, some guidance is given from this expression in order to calculate the cell size elutriated at any flow rate. For the experiments, the JE-6B elutriation system with appropriated rotor and standard chamber was used. The standard chamber has a volume of 4.2 ml. Human peripheral blood mononuclear cells were isolated from blood on Ficoll-Paque gradients. The mononuclear cells (300–500 x 10⁶), suspended in 35 ml RPMI complete medium, were loaded into a Beckman elutriation centrifuge. Separation by counterflow centrifugal elutriation was performed at a constant rotor speed from (3,000 rpm) and a variable flow rate (6-36 ml/min) at 4°C. Phosphate-buffered saline (PBS^def) supplemented with 0.1% bovine serum albumin and 0.01% EDTA was used as an elutriation medium. Cells were drawn in the separation chamber at flow rates of 7 ml/min, following flushing at 15 ml/min in order to remove trombozytes and erythrocytes. The

lymphocyte and NK-cell population was eluted at flow rates of 28.5 ml/min, whereas the monocytes population was eluted at flow rates of 36 ml/min.

The purified cell population was centrifuged at 1,400 rpm for 10 min at 4°C and resuspended in culture medium.

4.1.9.4. Isolation of untouched monocytes

After elutriation, the monocytes population had a purity of 60-80%. Therefore, the cells had to be further purified to get a homogenous monocyte population by using a magnetic activated cell sorting (MACS) kit, "Monocyte Isolation Kit II (human)". All non-monocytes were indirectly magnetically labeled by using a cocktail of biotin-conjugated antibodies as the primary label reagent, followed by anti-biotin monoclonal antibodies conjugated to microbeads as a secondary labeling reagent. The cell suspension was loaded onto a MACS LS column that was placed in the magnetic field of a MidiMACS separator. The magnetically-labeled non-monocytes were retained on the column, whereas all cells that were not labeled passed through and could be collected as the purified and untouched monocyte population. The separation was performed as described in the manufacturer’s manual. The purity of the cultures was analyzed via FACS analysis (4.7) using antibodies against CD14, CD11b and BDCA2 and usually achieved 86-96% purity.

The cell number was counted (4.1.6), and monocytes were seeded in medium with 4% AB at 3 x 10⁶/ml in 96-well flat-bottom plates and could be used for further experiments (4.6/4.8). As a control, PBMC were also used for stimulation (0.3x10⁶ cells/well).