Cell culture insert

The invasion assay was performed in 24-well plate for 20 h. The transfected cells (3 × 10⁴) in 200 μL of serum free medium were seeded onto upper Matrigel matrix (Corning, New York, NY, USA) coated Cell Culture Insert (Greiner Bio One, Kremsmünster, Austria). The lower chamber contained 900 μL of complete culture medium. The invaded cells were fixed with methanol for 10 min and stained with 0.005% crystal violet for 1 h at room temperature. The numbers of invaded cells were counted under the microscope from 10 random fields^{55 (link)}.

To localize EST6 in the antenna, we performed immunohistochemistry with the anti-EST6 antibody above on transgenic flies expressing RFP under the control of either the Orco or lush promoter or GFP under the control of the elav promoter. Est6 null mutant flies were used as a control for the specific labelling of the antibody. Specifically, heads with antennae still attached from 5-day-old Orco^Gal4/UAS-mCD8::RFP, elav^LexA/LexAOP-mCD8::GFP, lush^Gal4/UAS-mCD8::RFP or Est6 null mutant males were fixed for 3 h in 4% paraformaldehyde with 0.2% Triton X-100, then washed for 1 h with PBS containing 0.2% Triton X-100 (PBST). The heads were then embedded in Tissue-Tek^TM (CellPath) and cryosections (15 μm) were set in cell culture insert (Greiner Bio-one). After blocking with 3% normal goat serum and 1% BSA in PBST (1 h at room temperature), the anti-EST6 antibody was diluted from 1:3,000 to 1:750 (v:v) in the blocking solution (3% normal goat serum in PBST) and incubated overnight at room temperature. After a brief rinse in PBST, an anti-mouse conjugated Alexa-488 or Alexa-596 (Invitrogen) was applied at a concentration of 1:800 (v:v) in the blocking solution for 4 h at room temperature. Tissues were mounted in Slowfade reagent with DAPI (Invitrogen). Images were captured on a Leica SP5 confocal microscope and analysed using ImageJ 1.47 v (http://imagej.nih.gov/ij).

The migration and invasion assays were performed in 24-well plate for 16 and 20 h respectively. 3 × 10⁴ cells in 200 μL of serum free medium were seeded onto upper Cell Culture Insert with 8 μm pores (Greiner Bio One, Kremsmünster, Austria) for migration assay and Matrigel matrix (Corning, New York, NY, USA) coated Cell Culture Insert for invasion assay respectively. The lower chamber contained 900 μL of complete medium. The migrated and invaded cells were fixed with methanol for 10 min and stained with 0.005% crystal violet for 1 h at room temperature. The numbers of migrated and invaded cells were counted under the microscope from 10 random fields.

Low-density cells seeded in a 12-well plate were co-cultured with high-density cells plated in a Transwell insert (Thincert cell culture insert, Greiner, 665640). The surface of the Transwell insert is a translucent PET membrane with a pore size of 0.4 μm; this allows for the diffusion of soluble factors from the Transwell insert to the 12-well plate located underneath. The 12-well plates were coated with fibronectin at a concentration of 5 μg/cm². Coatings are not required for Transwell inserts. However, it is necessary to condition the Transwell inserts by covering the entire surface of the membrane with the cell culture medium prior to cell seeding, in order to improve the attachment and spreading of cells. The cells were seeded at a low density of 5 × 10³ cells/cm² in the 12-well plate or at a high density of 2 × 10⁵ cells/cm² in the Transwell insert, one day prior to differentiation. The medium volume was 1.5 mL for the 12-well plates and 1 mL for the Transwell inserts. Furthermore, 4 h after plating and once the cells were attached to the surface, co-culturing was commenced. Cells were induced using 3 μM CHIR and cultured under the protocol previously established for low density differentiation^{14 (link)}. Fourteen days after induction, the cells were harvested for evaluating the cTnT expression via flow cytometry.

Indirect co-culture experiments were performed using semipermeable cell culture inserts with 8 µm pores for 12-well plates (Greiner Bio-One, Frickenhausen, Germany). Osteoblasts were seeded in cell culture inserts with 50,000 cells per insert in DMEM medium supplemented with additives, as mentioned in 2.1. After 24 h of incubation, the inserts were transferred to cell culture plates containing 300.000 OLCs per well. The cells were stimulated with IL-6 (50 ng/mL), recombinant human sIL6R (50 ng/mL), and sarilumab (250 nM) for seven days. Since it was shown that the supplementation of the different cell culture additives, such as M-CSF or dexamethasone, affects the differentiation of the cell types, only ascorbic acid and glutamine were added to the media for these stimulation experiments [18 (link)]. RPMI medium used for stimulation was supplemented with 2% FCS and 2% stable glutamine, while 10% FCS and 50 μg/mL ascorbic acid was added to DMEM medium. Indirect co-culture experiments were performed for seven days. Single cultures of human osteoblasts or OLCs under the same conditions served as controls. For the following analyses, the two cell types were examined separately, whereby the secretion of proteins was determined in the total supernatant of both cell types.