Axiovision z1

Recombinant human matrilin-2 protein was purchased from R&D systems (Minneapolis, MN), coated onto 22 mm tissue glass coverslips at 20ug/ml as described previously (Malin et al., 2009 (link)) and incubated overnight. The agarose migration assay was performed as previously described (Chernousov et al., 2001 (link)). The agarose for the drop migration assay was diluted to a 0.03% concentration. After 24 h of incubation, the cells were visualized under phase-contrast microscopy.
For cellular adhesion, SCs were placed atop matrilin-2 coated plates and incubated for 24 h. Cells were then fixed at room temperature with 4% paraformaldehyde for 10 min. Subsequently, cells were washed with PBS, followed by permeabilization for 30 min using 0.1% Triton X-100. Cells were stained with rhodamine phalloidin (Cell Signaling Technology, Danvers, MA) for immunofluorescent imaging. After 30 min, the cells were washed three times with PBS and incubated in 4′,6′-diamidino-2-phenylindole dihydrochloride (1:1,000; Sigma-Aldrich, St. Louis, MO) for 5 min at room temperature to counterstain the cell nuclei. The specimens were washed three times with PBS, each for 5 min, and were then mounted with an antifade solution. Immunolabeled cells were examined via fluorescence microscopy (Zeiss Axiovision Z1, Oberkochen, Germany). All experiments were carried out in triplicate.

PKM2 subcellular localization was detected by immunofluorescence. After 24 h of attachment in cell chamber slides (Millipore), cells were treated with OXA and fixed to coverslips in cold acetone for 10 min at room temperature. Blocking and permeabilization was done with PBS-T/ FBS 10%. Cells were incubated at room temperature with a rabbit polyclonal anti-PKM2 primary antibody (Cell Signaling; 1:100) for 1.5 h and subsequently, with secondary antibody anti-rabbit Alexa-568 (Invitrogen; 1:200). Nuclei were stained with DAPI gold-antifade reagent (Invitrogen). Coverslips were observed with a fluorescence microscope Axiovision Z1 by using Apotome system at 40x immersion oil lens (Carl Zeiss, Heidelberg, Germany). Multiple images were taken at different focus distances by using z-stacking (thickness interval: 0.750–1 μm) to localize PKM2 at different focal depths.

Pictures of DAPI-stained chromosomes or differentiated cells were photographed under a Zeiss Axiovision Z1 fluorescent microscope with 20× and 63× objectives, and analyzed with Axiovision 4.8 software. Morphology of vASC cultures and ASC.B6 cells was checked by Olympus CKX41 inverted light microscope with 4× objective and photomicrographs were taken with an Olympus Camedia C-5060 camera (Olympus Holding Europa GmbH).

In total, 25 mmol of rhodamine 123 was added to the medium of cysts derived from cultured cells and incubated for 5 min. Some of them were incubated with 100 μM verapamil for 30 min before adding the dye. After washing with PBS 3 times, the 3D culture media were added and incubated for 40 min at 37 °C. Images were acquired with a Floid^TM Cell Imaging Station (Life Technologies) and AxioVision Z1 microscopy (Carl Zeiss, Oberkochen, Germany).