2.2.5.1 3D-CoG spleen slice infection model (3D-CoG/SSIM)
Collagen gels (3D-CoG) were generated as described previously (Freundet al., 2008) and incubated without agitation. Bacteria were prepared as described in chapter 2.2.1.4 (p. 30) and subsequently suspended in liquid collagen solution (about2x104/ml). Liquid collagen solution consisted of 1.78 mg/ml bovine type I collagen (Purecol, Advanced Biomatrix) in RPMI 1640 medium adjusted to pH 7.4. 10 µl of this solution were dispersed on the bottom of a microscopic dish (9.4 x 10.7 mm, µ-slide 8 well, ibidi). The samples were allowed to polymerize for 45 min (37 �; 5 % CO2). 3D-CoGs were overlaid with 150 µl cell culture
medium (RPMI 1640).
For neutrophil challenge of microcolonies in 3D-CoG, native spleens from 8-12 weeks old heterozygous lys-EGFP mice (Faust et al., 2000) were used. Mice were sacrificed by CO2 asphyxiation and spleens were harvested and cut into 300 µm slices with a vibrating
blade microtome (Leica) at 4 �. Supernatants from 3D-CoG samples were removed and then 3D-CoG were overlaid with spleen slices. To immobilize the slices on the 3D-CoG, a drop of 4 % NuSieve GTG agarose (Lonza) was applied. After solidification, 150 µl of RPMI 1640 were added.
The manual process of overlaying 3D-CoG with tissue slices can partially result in compression or injury of some areas of the collagen gel. To compensate for artifacts, only microcolonies in a lateral distance of about 200 µm to the tissue slice-collagen gel interface were analyzed after verifying that the collagen gel in this area was not compressed or in- jured. This was achieved by visualization of the 3D-CoG structure with confocal reflection microscopy. The samples were incubated at 37 �, 5 % CO2 and saturated humidity in a
For experiments withS. aureus, the medium for overlaying 3D-CoG was supplemented with 3 mg/ml fibrinogen from human plasma (Calbiochem) (3D-CoG/Fib). Optionally, argatroban (Santa Cruz) was added to the growth medium at the concentrations mentioned in the text.
2.2.5.2 3D-CoG migration assay
LPS-stimulated dendritic cells were infected with Y. enterocolitica and their ability to migrate after infection was analyzed in custom-built migration chamber (Fig. 2.1). 6x105 DCs were infected for 30 min at an MOI of 20 with the respective Yersinia strains in a volume of 200 µl RPMI/10 % FCS in 24 well plates. After infection, PBS with 20 µg/ml gentamicin was added to the wells, the cell supension was transferred to 1.5 ml tubes and cells were washed two times with 1 ml PBS/Genta. Cells were resuspended in 100 µl RPMI/10 % FCS and incorporated in 3D-CoG in a custom-built setup (final cell concentration in 3D-CoG ca. 7 x 105 DCs/ml). After polymerization of the 3D-CoG, a CCL19 gradient was established by applying 20 µl 0.6 µg/ml CCL19 (Peprotech, Hamburg) to the open side of the 3D-CoG. Cell migration was recorded with a microscope at 37 �, 5 % CO2 for 3 h.
Figure 2.1: 3D-CoG migration assay setup. 6 holes (ca. 6 mm in diameter) were drilled in the bottom of a 35 mm cell culture dish. To the bottom they were closed by attaching a coverslip. On top, ca. 70 % of the hole were covered with a broken coverslip. Collagen solution was applied with a pipette and the setup was tilted 90 degrees so that the collagen sank to the side of the well opposite of the opening. After polymerization, chemoattractant solution (CCL19) was applied to the residual space in the well, establishing a CCL19 gradient in the 3D-CoG. Cells migrated towards the gradient in 3D-CoG.
2.2.5.3 Under-agarose migration assay
The under-agarose migration assay (Fig. 2.2) provides a setup for quantitative and qual- itative analysis of cell migration and was conducted according to Heit and Kubes (2003) and Lämmermannet al. (2009). In the present work it has been used to characterize the effects ofYersiniaeffector proteins on migratory behavior of single dendritic cells. 5 ml 2x HBSS buffer and 10 ml RPMI 1640/20 % FCS were mixed and heated to 68 � in a water bath. In a separate tube, 0.125 g ultra-pure agarose was dissolved in 5 ml H2O by boiling
in a microwave. Both solutions were combined and 1.8 ml were pipetted into custom- made chambers composed of a ring cut from a 50 ml Falcon tube fixed to a small petri dish. After solidification of the 0.625% agarose, wells were punched in the gel (ca. 2 mm in diameter). Gels were equilibrated for 1 h at 37 �; 5 % CO2. Usually a central well
(attractor well) was filled with 5 µl chemoattractant (1.2 µg/ml CCL19; Peprotech, Ham- burg) and in a distance of 5 mm 1-3 wells were punched (responder wells). The chemokine diffuses through the agarose and thereby a chemokine gradient from the attractor to the responder wells is created. The responder wells were filled with 5 µl DC suspension (105
cells/well). Alternatively, cells directly injected under the agarose for transfected cells or if initial cell migration from the responder well under the agarose was inhibited, e. g. due to pre-infection. Bacteria were prepared as described in in chapter 2.2.1.4 (p. 30) and injected under the agarose with a fine pipette tip in 0.5 µl.
Figure 2.2: Under-agarose migration assay setup. 0.625 % agarose was poured in a well constructed by a section of a 50 ml Falcon tube. After solidification of the agarose, small wells were punched in the gel. Usually, a central well was filled with chemoattractant (CCL19) and acted as the attractor well and wells in the periphery were filled with dendritic cells in solution. A gradient is established outward from the attractor well, causing the DCs to squeeze underneath the agarose and migrate towards the gradient.
2.2.5.4 Ex vivo microscopy in the organ chamber
The organ chamber (Fig. 3.34 A, B, p. 87) was conceived by A. Wieser, designed and optimized by A. Wieser and C. Guggenberger and machined from aluminum and anodized by A.Wieser and Dipl. ing. Michael Achtelik. It is suitable for standard cross tables for ISO 8037/I microscope slides (76 x 26 mm) and consists of four main parts which are held together by screws. The chamber is sealed water and gas tight against the environment by two glass coverslips (32 x 24 mm) and can be used on both inverted and upright microscope setups. All connectors of the organ chmaber are in the Luer-Lock system to allow usage of standard medical infusion tubing. Gas composition is adjusted by a custom-built mem- brane oxygenation device (Fig. 3.34 C, p. 87, constructed by A. Wieser) and an active gas supply system (The Brick, Life Imaging Services, Basel). Oxygen concentration and pH values in the medium were measured with PSt3 Oxygen Optodes (PreSens, Regensburg, Germany) and a pH electrode (320 pH-Meter, Eppendorf, Hamburg), respectively.
Ex vivo cultivation of organ explants was performed in DMEM (Sigma-Aldrich, Ham- burg, Germany) supplemented with 10 % fetal calf serum (Invitrogen). Ex vivo infection in the organ chamber was performed with E. coli NU14, S. enterica and E. histolytica, respectively; intravesical in vivo infection was performed with E. coli NU14 as described in chapter 2.1.6.3 (p. 29). Bacterial counts were determined by photometric measurements and subsequent determination of cfu.
E. coliNU14 About3x108 cfuin 30 µl PBS were injected into the apical compartment of the urinary bladder explant in the organ chamber.
S. enterica About 2 x105 cfu in a volume of 50 µl PBS were injected into the apical compartment of the gut explant inside the organ chamber.
E. histolytica 3.8 x 104 cfu amoebae in a volume of 50 µl PBS were injected into the apical compartment of the organ chamber containing the caecum explant and incubated for 20 min before starting perfusion.