Cell biological and virological methods

2.2.2.1 Cell culture

Passaging of cell lines

Cell lines were kept in specific growth media (see 2.1.7) and cell culture flasks (T25 or T75) in incubators at 37 °C and 5 % CO2.

Adherent growing cells were trypsinated using Trypsin/EDTA and split every 3-4 days at various ratios from 1:4 to 1:10 depending on the growth rate of

Cell lines growing in suspension were split directly.

BSR-T7/5 cells were supplemented with 2 mg/mL G418 (Geneticin) every other passage. U3A3 cells were supplemented with 2 mg/mL G418 (Geneticin) and 0.5 mg/mL Hygromycin every other passage.

Seeding of cells

Adherent growing cell lines were trypsinated and resuspended in growth medium. Cell numbers/mL were calculated based on estimated cell numbers/flask (T25: 3x106_{; T75: 9.4x10}6_{) or determined using a Neubauer}

counting chamber.

Cells were seeded in various culture dishes as follows:

dish number of cells total volume of growth media 10 cm² dish 1.5x106 _{10 mL} 6 cm² dish 6x105 _{2-5 mL} 3.5 cm² dish 3x105 _{2 mL} 6-well 3x105 _{2 mL} 12-well 2x105 _{0.5-1 mL} 24-well 1x105 _{300-500 µL} 48-well 5x104 _{200-400 µL} 96-well 1-2x104 _{100-200 µL}

2.2.2.2 Isolation and cultivation of bone marrow derived cells (BMDCs)

Femur and tibia were isolated from hindlimbs of C57/BL6 mice and bone marrow was extracted by extensively flushing with 1x PBS. Bone marrow was passed through a 40 µm cell strainer using 1x PBS and the cell suspension was centrifuged (400 g; 4°C; 7 min).

The supernatant was discarded and the cell pellet was resuspended in 2 mL of 1x ACK and incubated at R.T. for 2 min (erythrocyte lysis). 28 mL of 1x PBS

were added to stop the lysis process and the cells were pelleted again by centrifugation (400 g; 4 °C; 7 min).

The pellet was resuspended in 10 mL of 1x PBS and the cell number was determined using a Neubauer counting chamber. 2-3x106 _{cells/mL were}

cultivated in T25 cell culture flasks.

To stimulate differentiation of BMDCs into pDCs, 20 ng/mL of human recombinant Flt3-L as well as 50 µM of β-mercaptoethanol were added to the cell culture and cells were incubated at 37 °C and 5 % CO2 for 7 days.

2.2.2.3 Transfection

Cells were transfected with DNA, RNA or peptides using Lipofectamine 2000

(Invitrogen) or calciumphosphate method (Mammalian Transfection Kit; Stratagene) according to the manuals.

For co-immunoprecipitation assays, a solution containing 1 µg/µL PEI instead of Lipofectamine 2000 solution was used following the Lipofectamine 2000

protocol.

2.2.2.4 Infection assays

According cell numbers were seeded in multiwell plates and directly infected in suspension with calculated MOIs of rapidly thawed virus stocks.

2.2.2.5 Generation of MV stocks

Two confluent T75 flasks of Vero cells were harvested and pelleted by centrifugation (1,600 rpm, 4 °C, 10 min). The supernatant was discarded, cells were resuspended in 500 µL of growth medium and infected with MV to an MOI = 0.25. The total volume was adjusted to 2.5 mL and the suspension was incubated at 37°C for 1 h with shaking gently every 10 min. Afterwards, the

infected cells were seeded equally in 6 T75 cell culture flasks with a total of 16 mL growth medium/flask and incubated at 37 °C for 40-48 h.

As soon as most cells had formed numerous small syncytia, the cells were frozen at -20 °C. Cells were thawed on ice and lysates were cleared from cell debris by centrifugation (1,600 rpm, 4 °C, 10 min). Cleared lysates were aliquoted and stored at -86 °C.

2.2.2.6 Generation of RV stocks

One confluent T25 flask of BSR-T7/5 cells was split 1:4, the cells were infected with RV at an MOI = 0.1 and incubated at 37 °C.

The supernatant was collected 72 h p.i. (1st _{harvest), cleared from cell debris by}

centrifugation (1,600 rpm, 4 °C, 10 min), aliquoted and stored at -86 °C. Cells were supplied with fresh growth medium and incubated at 37 °C.

A 2nd _{harvest was performed analogous to the first harvest 144 h p.i.}

2.2.2.7 Generation of recombinant RV (helper virus free)

BSR-T7/5 cells were seeded in 6-well plates 16 h prior to transfection. Growth medium was replaced with D-MEM 1 h prior to transfection.

10 µg of pSAD (RV genome encoding plasmid) and 5 µg pTIT-RV-N, 2.5 µg pTIT- RV-P and 2.5 µg pTIT-RV-L helper plasmids were transfected using the calciumphosphate method. The cells were washed twice with growth medium, supplied with 2 mL of growth medium 3.5 h p.tr. and incubated at 37 °C for 72 h.

The supernatant was collected 72 h p.tr., cell debris was removed by centrifugation (1,600 rpm, 4 °C, 10 min) and put on freshly seeded BSR-T7/5 cells in 6-wells. These were incubated at 37 °C for 72 h (Supernatant passage 1A). The transfected cells were supplied with 2 mL of fresh growth medium and incubated at 37 °C for another 72 h.

The supernatants of the transfected cells were collected again 144 h p.tr. and treated as before (Supernatant passage 1B). The transfected cells were trypsinized and 25 % of the cells were seeded in new 6-wells (Split), whereas 75 % of the cells were seeded in T25 cell culture flasks and incubated at 37 °C for further 72 h.

All 6-wells (Supernatant passage 1A, 1B; Split) were analyzed by IF using acetone fixation and FITC-labelled anti-N antibody (see 2.2.3.7) for the formation of viral foci.

The supernatants of virus containing T25 cell culture flasks were harvested, cell debris was removed by centrifugation (1,600 rpm, 4 °C, 10 min), supernatants were aliquoted and stored at -86 °C. Viral titers were determined as described in 2.2.2.8.

2.2.2.8 Titration of virus stocks

In case of MV, Vero cells were used for titration of virus stocks, in case of RV, BSR-T7/5 cells were used.

2x104 _{cells were seeded into 96-wells to a total volume of 100 µL/well and}

incubated at 37 °C for 2 h.

500 µL of virus stocks were thawn on ice and serially diluted 1:10, resulting in dilution series from (100 _{[=undiluted] to 10}-8_).

100 µL of each dilution was added to one well in duplicates and cells were incubated at 37 °C for 48 h.

Cells were fixed using the PFA/NH4Cl (for MV) or acetone (for RV) protocol (see

2.2.3.7). Cells were stained with diluted FITC-labelled anti-N antibodies (MV: Millipore; RV: FDI) and incubated either at 4 °C (MV) or 37 °C (RV) for 2 h. Foci were counted from each well using a UV microscope (Olympus) and foci forming units per mL (ffu/mL) were calculated from these results.