Animals used for the experiments were C57BL/6 from the MEZ Leipzig and Charles River (Sulzfeld, Germany). They were handled in accordance to local animal ethics regulations.
Bone marrow isolation and culture of NA-BMC
Femurae and tibiae of 2–3 month old C57BL/6 mice were isolated, opened and centrifuged to obtain bone marrow. 107 bone marrow cells were cultivated for 24 h in a 60 mm petri dish and in 10 ml Dulbecco’s modified eagle medium (low glucose) (DMEM, Hyclone Laboratories Inc.), supplemented with 10% fetal calf serum (FCS) (Invitrogen), 10-8 M dexamethasone and 100 units/ml Penicillin/Streptomycin (Invitrogen). After 24 h the non-adherent cells were flushed off and transferred to a new dish (protocol 2; Figure 1). This 24 h adhesion period was repeated 4 times to derive NA-BMC cells (protocol 1; Figure 1).
The non-adherent cells of day 1 (classical replating protocol to derive macrophages, protocol 2) and NA-BMCs from day 4 (protocol 1) were resuspended in 5 ml osteogenic medium (DMEM, 10% FCS, 10-8 M dexamethasone, 50 μg/ml ascorbic acid) in a 60 mm dish. Every 3 days, the medium was changed. After 10 days, the cells were fixed with cold ethanol and alkaline phosphatase (ALP), calcium (Alizarin red), collagen (Sirius red) and methylene blue (total colonies) staining performed. The colony numbers were determined using the program ImageJ.
The cells in 60 mm petri dishes were fixed with cold ethanol for 15 min. They were washed with tap water. Tris (200 mM, pH 8.5) was mixed with naphthol phosphate ASBI (50 μg/ml) and fast red (1 mg/ml) (Fast red was always mixed fresh). 5 ml of the mixture was added to petri dishes. The dishes were shaken for 2 h at room temperature. Afterwards they were washed with tap water and allowed to dry. Photographs of the dishes were taken and colony numbers determined with ImageJ.
Alizarin red staining (Calcium)
Cells were fixed for 15 min with ice-cold ethanol, afterwards washed with tap water. 5 ml of a solution of 1 mg/ml alizarin red in distilled water, pH 5.5 were added. The petri dishes were shaken for 2 h, afterwards washed with tap water and allowed to dry. Images were analyzed for calcium amount using ImageJ.
Sirius red staining (Collagen)
Cells were fixed in ice-cold ethanol for 15 min and washed with tap water. 1 mg/ml sirius red was solved in picric acid. 5 ml of the mixture was added to the cells and the petri dishes shaken for 18 h at room temperature. Then the cells were washed with tap water till red color was completely eluted. The dishes were photographed and analyzed for the amount of collagen (ImageJ).
Methylene blue staining (total colony numbers)
Cells were fixed in ice-cold ethanol for 15 min. They were washed in tap water. 1 mg/ml methylene blue was solved in 10 mM borate buffer, pH 8.8. 5 ml of the mixture was added to the petri dishes and shaken for 30 min. The dishes were washed with tap water until all dye was eluted, photographed and analyzed using ImageJ.
Astrocyte conditioned medium
Astrocyte conditioned medium was produced by incubating medium (DMEM/10% FCS) 24 h with primary astrocyte cultures produced as described by Sievers .
Differentiation towards microglia-like cells
On day 1 (protocol 2) and day 4 (protocol 1), the non-adherent cells were flushed off and transferred to a new dish. Afterwards cells were differentiated for 6 days in 10 ml DMEM/10% FCS, 50% ACM and 20 ng/ml granulocyte-monocyte colony stimulating factor (GM-CSF). Controls were cultured only in DMEM/10% FCS.
Cell were stained for F4/80 (AF488 labelled, eBioscience) (1:250), CD11b/CD45 (AF488 and PE labelled eBioscience) (1:250 and 1:100) and CD34 (PE labelled, Caltag Laboratories) (1:100), CD45 R (RPE labelled, Southern Biotech) (1:100). Cells were trypsinized, centrifuged (300 g for 5 min) and fixed (4% paraformaldehyd). The fixed cells were washed with PBS, incubated for 2 h at 4°C with primary antibody, washed and analysed in a Beckmann Coulter FC 500 Flow cytometer.
3*105 cells were activated with 0.1 μM phorbol myristic acid for 15 min. Then they were incubated in 50 μl DMEM/10% FCS together with 50 μl 1:10 diluted opsonised beads (2.25*107 beads) (Sigma) for 48 h at 37°C, 5% CO2. Cells were trypsinized and resuspended in DPBS (Invitrogen) and fluorescence was measured in a Beckmann Coulter FC 500.
3*105 cells were activated for 15 min with 0.1 μM PMA and controls without PMA. Activated and control cells were incubated with 50 μM DHR123 for additional 15 min. Afterwards the cells were fixed with 4% PFA and fluorescence was measured in a Beckmann Coulter FC 500. Oxidative burst was defined as signal to noise ratio (The ratio of fluorescence of activated to control cells).
Living brain slice cultures
Brains from 2–3 month old C57BL/6 mice were transferred to cold Hank’s buffered salt solution (HBSS)/10% FCS (both Invitrogen). A VT 1000 S vibratome (Leica) was used to cut the brain in 350 μm slices. The slices were culture on a membrane (Millicell CM 0.4 μm, Millipore) at the liquid air interface of medium consisting of 50% DMEM/high glucose (HyClone Laboratories Inc.), 25% horse serum (Invitrogen), 25% HBSS (Invitrogen), 1 μg/ml insulin (Invitrogen), 100units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). The brain slices were cultured for 9 days and the viability of the brain slices was assessed using DAPI/propidium iodide staining. For analysis the slices were scanned using a confocal microscope (TCS SP2, Leica Microsystems).
Invasion of living brain tissue
On day 9 of brain slice culture, differentiated cells were treated with DIO (Invitrogen) for 20 min, washed and seeded on the top of the brain slices. A plastic ring was used to keep the cells from flowing off the slices. Cells and brain tissue were co-cultivated for additional 10 days . After 10 days migration of cells into brain tissue was measured by scanning the slices with a confocal microscope (TCS SP2, Leica Microsystems) to a depth of 160 μm.
Data is presented as means ± SE. SigmaPlot 10.0/SigmaStat 3.5 software (SYSTAT, Erkrath, Germany) was used to perform statistical analysis. For comparison of different groups ANOVA was used.