Lab tek 2 chambered coverglasses

Mitochondrial morphology and number of lysosomes were analyzed by live cell microscopy. For this, MEF were cultured in Lab-Tek^®II chambered coverglasses (Nalge Nunc International). Lysosomes were stained with 50 nM LysoTracker^® Red DND-99 and mitochondria with 200 mM MitoTracker^® Green FM or MitoTracker^® red FM (all Invitrogen) for 15 min at 37°C. Analysis of the number of lysosomes and/ or mitochondrial morphology was performed using an inverted Zeiss Axiovert microscope (Zeiss Plan-Apochromat 63 x/1.4) with an incubation chamber temperature of 37°C. Time lapse pictures were taken with an AxioCamMRm camera (Zeiss) in 5 s intervals for up to 5 min using AxioVision software (Zeiss). The series of pictures was saved uncompressed and analyzed with AxioVision software (Zeiss). Colocalization studies, MEF cells were incubated with 250 nm MitoTracker^® red FM for 30 min and afterward fixed with 4% paraformaldehyde for 20 min. After blocking of fixed cells with 10% normal serum, cells were stained with cytochrome C (1:500, Cell Signaling) overnight and the secondary antibody anti-Cy2 (1:300, Jackson ImmunoResearch) for 2 h. DAPI (Invitrogen) was used to stain nuclei. Images were analyzed by Zeiss LSM 510 Confocal Microscope and Zeiss LSM Image Examiner LSM510.

MAECs (4 × 10⁴ cells/well) were seeded onto Nunc Lab-Tek II Chambered Coverglasses and incubated overnight in a CO₂ incubator. Then, PEG-modified or unmodified C3H10T1/2/GFP cells (1 × 10⁴ cells/well) were seeded onto monolayered MAECs. The culture medium was removed 2 h after incubation, and the cells were incubated with 4% paraformaldehyde solution. Thirty minutes after incubation, 0.2% Triton X-100 in PBS and 1% bovine serum albumin in PBS were added to achieve permeabilization and blocking, respectively. The fixed cells were then incubated with anti-phospho-focal adhesion kinase (p-FAK) IgG primary antibody for 1 h at room temperature and stained with tetramethylrhodamine isothiocyanate (TRITC)-labeled secondary antibody for 1 h at room temperature. After immunostaining, the samples were mounted with Fluoromount-G and observed using a TCS P8 confocal laser scanning microscope. The fluorescence intensity of phospho-FAK observed in stained C3H10T1/2/GFP cells was measured by region of interest (ROI) analysis. Then, the average pixel intensity (pixel intensity per cell) of 15–20 randomly selected C3H10T1/2/GFP cells was calculated.

Fc-receptors on THP-1 cells were blocked with 1% human serum (HS, from SIGMA) in medium for 5 min at RT, followed by sub-labeling with non-blocking TS2/4-Atto647N in serum free/phenol-red free medium for 5 min at RT. As we were in particular interested to track LFA-1 nanoclusters instead of single LFA-1 heterodimers, we chose whole antibody over Fab fragments for tracking. Cells were extensively washed in serum-free/phenol-red free medium. After washing, cells were seeded for 10 minutes at 37 °C on potassium hydroxide cleaned and sonicated Labtek II chambered cover-glasses (4 well, Nunc), coated with 20ug/ml fibronectin (Roche). Treatment with SMase or myriocin was performed prior to imaging and the drugs remained present for the time of the experiment. CytoD was added to the imaging medium and incubated for 5 min 37 °C prior to imaging.