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For washing and harvesting, mammalian cells were centrifuged using the general cell program: 8 min, 300×g, 4°C.

4.1.1 Cell line culture conditions and passaging

Cells were cultured in RPMI 1640 (HyClone) or DMEM (Gibco) routinely supplemented with 10% inactivated FCS, L-glutamine (2 mM), sodium pyruvate (1 mM), antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin), 2 ml vitamins, non essential amino acids and 50 µM ß-mercaptoethanol. Media supplements were purchased from Gibco and Biochrome (L-glutamine), respectively.

FCS was heat inactivated for 30 min at 56°C before use. Exceeding incubation times and higher temperatures should be avoided because heat sensitive growth factors could be damaged. Each batch of FCS as well as each RPMI batch was tested before use.

Culturing of cells was performed at 37°C, with 5% CO2 and 95% relative humidity in an

incubator. U937, THP-1 and KG-1 cells grow in suspension and were split 1:4 to 1:8 in fresh medium every 2-4 days.

4.1.2 Culturing of stably transfected Drosophila S2 cells and

expression of the methyl binding polypeptide MBD-Fc

MBD-Fc stands for a fusion protein consisting of the methyl-CpG binding domain (MBD) of human MBD2 (methyl-CpG binding domain protein 2) and the Fc-tail of human IgG1. The MBD-Fc vector was stably transfected into Drosophila S2 cells using the Effectene transfection reagent (Qiagen) and hygromycin selection. A detailed description of design and generation of the fusion protein can be found in Gebhard, 2005 and Gebhard et al., 2006b.

Expansion in cell culture bottles

MBD-Fc S2 cells were seeded at a density of 1-2×106 cells/ml in Insect-Xpress medium (Lonza) including 50 U/ml penicillin and 50 µg/ml streptomycin but without FCS at 21-23°C.

400 µg hygromycin was added for selection of plasmid containing cells. Cells were splitted once a week, without exceeding 10×106 cells/ml.

Protein production

Cells were transferred into 2,000 ml roller bottles and cultured at a density of 4×106 _cells/ml

in up to 400 ml Insect-Xpress medium supplemented with penicillin, streptomycin and hygromycin as described above. Cells should not exceed a density of 10×106 _{cells/ml. For}

large-scale protein production, after 3-5 days the culture media was exchanged and 5×106 _{cells/ml were seeded in 400 ml Insect-Xpress medium. Instead of hygromycin, 0.5 mM}

CuSO4 was added to stimulate the metal inducible promoter of the used vector. The MBD-Fc

containing culture medium was harvested after 4 days like described in section 4.2.1. For recovery, cells were cultured again in Insect-Xpress medium containing standard antibiotics and selection antibiotic for 3-5 days. The cycle of production was repeated until protein quality and amount clearly decreased.

4.1.3 Assessing cell number and vitality

The number of viable and dead cells was determined by Trypan blue exclusion. Cell suspensions were diluted with Trypan blue solution and then counted in a Neubauer haemocytometer. Dead cells appear blue since the blue stain is able to enter the cytoplasm. The concentration of viable cells was then calculated using the following equation:

Number of viable cells/ml C=N×D×104

N: average of unstained cells per corner square (1 mm containing 16 sub-squares) D: dilution factor

Required solutions and materials:

Trypan blue solution: 0.2% (w/v) Trypan blue in 0.9% NaCl solution Neubauer haemocytometer slide with coverslip

4.1.4 Freezing and thawing cells

Cells were harvested and resuspended at 5-10×106 cells/ml in 800 µl ice cold medium, including 10% FCS. After inverting the mix and transferring it into cryo-vials, 160 µl DMSO

(10% final) and 640 µl FCS (40% final) were added and the tubes were rapidly inverted to mix cells properly. To allow gradual freezing at a rate of 1°C/min, the cryo-vials were placed in isopropanol-filled cryo-containers (Nalgene) for two hours, then transferred to -80°C for 48 h. For long-term storage, samples were transferred in liquid nitrogen (-196°C).

4.1.5 Mycoplasma assay

Cell lines were routinely checked for mycoplasma contamination by the MycoAlert® Mycoplasma detection assay (Cambre, Rockland, USA) according to the manufacturer’s instructions.

4.1.6 Isolation of human monocytes

Peripheral blood mononuclear cells (PB-MNCs) were separated by leukapheresis of healthy donors (Graw et al., 1971), followed by density gradient centrifugation over Ficoll/Hypaque (Johnson et al., 1977). Monocytes were then isolated from MNCs by counter current centrifugal elutriation (Sanderson et al., 1977).

Elutriation was performed in a J6M-E centrifuge equipped with a JE 5.0 elutriation rotor and a 50 ml flow chamber (Beckman, Munich, Germany). After sterilizing the system with 6% H2O2

for 20 min, the system was washed with PBS. Following calibration at 2,500 rpm and 4°C with Hanks BSS, MNCs were loaded at a flow rate of 52 ml/min. Fractions were collected and the flow-through rate was sequentially increased according to Table 4-1.

Table 4-1 Elutriation parameter and cell types

Fraction Volume (ml) Flow rate (ml/min) Main cell type contained

Ia 1000 52 platelets

Ib 1000 57

B- and T- lymphocytes, NK cells

IIa 1000 64

IIb 500 74

IIc 400 82

IId 400 92

III 800 130 monocytes

Monocytes represent the largest cells within the MNCs and are therefore mainly obtained in the last fraction. Monocytes were >85% pure as determined by morphology and CD14 antigen expression. Low amounts of monocytes may be also detected in the IId fraction. Monocytes (fraction III) were centrifuged (8 min, 300×g, 4°C), resuspended in RPMI culture medium and counted. Monocyte yields were donor-dependent, typically between 10-20% of

total MNCs. Supernatants of monocyte cultures were routinely collected and analyzed for the presence of interleukin-6 (IL-6), which was usually low, indicating that monocytes were not activated before or during elutriation.