Axioimager

Astrocytes seeded onto eight-well Lab-Tek chamber slides were activated overnight with TNF-α (100 ng/ml). Primary encephalitogenic CD4⁺ T cells were isolated from spleens and lymph nodes of three EAE mice using the CD4⁺ magnetic beads (magnetic-activated cell sorting) selection kit (Miltenyi Biotec). CD4⁺ T cells were stained with 5(6)-carboxytetramethylrhodamine, succinimidyl ester (TAMRA) (10 μg/ml; Thermo Fisher Scientific) and 1 × 10⁶ cells were added per well in adhesion buffer (DMEM, 5% FCS, 2% l-glutamine, and 25 mM Hepes). CD4⁺ T cells were either untreated or preincubated for 15 min at RT with anti-mouse integrin α4 antibody (PS/2, 20 μg/ml; Pharmingen) or recombinant murine VCAM-1 (4 μg/ml; R&D Systems) before addition to the astrocytes. In some cases, astrocytes were incubated for 1 hour at RT with anti-mouse VCAM-1 antibody (M/K-2, 20 μg/ml; table S2) in adhesion buffer. CD4⁺ T cells were incubated with astrocytes for 30 min at RT on a shaker; three to four technical replicates per condition were used. Wells were washed, fixed with 1.25% glutaraldehyde, and analyzed for TAMRA⁺-adherent T cells using a Zeiss AxioImager; images were captured using a Hamamatsu ORCA-ER camera and analyzed using Volocity 5.2 software (ImproVision) and ImageJ.

Expression of fluorescent reporter proteins in individual cells was quantified as described before [6] (link) using fluorescence imaging on a AxioImager fluorescence microscope equipped with an ORCA AG CCD camera (Hamamatsu) or by flow cytometry on a FACScan (BD Biosciences). Wild-type level of CheY was estimated based on strain VS162. Expression of untagged CheR and CheB was quantified using immunoblotting with a respective polyclonal antibody as described previously [31] .

Immunofluorescence (IF) was performed on ice cold acetone-fixed cryosections (6 μm) using the following primary antibodies: polyclonal rabbit anti-mouse VE-cadherin Ab (Santa Cruz, Biotechnology, Dallas, Texas, USA), monoclonal rat anti-mouse PECAM-1 (CD31) Ab (BD Biosciences, Franklin Lakes, New Jersey, USA). Nonspecific binding sites were blocked with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, PA, USA) for 30 minutes. Thereafter sections were incubated with the primary antibody (1:200) for 1 h. Both incubations were performed in a humid chamber at room temperature. Slides were washed and the secondary antibodies (goat anti-rabbit Cy3 (Dianova, Hamburg, Germany) and donkey anti-rat alexa fluor 488 (Invitrogen, Carlsbad, CA, USA)) were applied overnight at 4 C (1:1000). For negative controls the staining procedure was performed as described without the primary antibodies. Data were acquired on a Zeiss AxioImager (20x/0.45 objective) equipped with a Hamamatsu ORCA–ER camera. Images were analyzed using Volocity software (ImproVision, Forchheim, Germany).