Confocal microscopy images (CZI files) of human chondrocytes in different cell culture media with stained nuclei and primary cilia

1. Methods

1.1 Cell culture

For investigating the influence of the cell culture medium composition on the lengths of primary cilia, human non-degenerative chondrocytes from a 30-year-old male donor (NHAC-kn, CC-2550; LONZA, Walkersville Inc., Walkersville, MD, USA) were used. These chondrocytes were seeded in passage four with a density of 28000 cells/cm² on collagen-coated glass coverslips (GG-15-Collagen; Neuvitro Corporation, Camas, WA, USA). The cells were cultivated in 12-well plates (Thermo Fisher Scientific Inc., Waltham, MA, USA) under hypoxic conditions at 37°C, 5% CO₂ and 5% O₂ with different media compositions for three days.
The basal medium consisted of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco™) including high glucose (GlutaMAX™), sodium pyruvate supplements (Thermo Fisher Scientific Inc., Waltham, MA, USA), as well as 1% penicillin/streptomycin (Pen/Strep; Thermo Fisher Scientific Inc.), 1% Amphotericin B (Biochrom GmbH, Berlin, Germany), and 50 µg mL⁻¹ ascorbic acid (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). To this basal medium, different supplements were added, creating four groups:
1) ITS: 1% Insulin-Transferrin-Selenium (ITS+™ Premix, BD Biosciences, Franklin Lakes, NJ, USA),
2) ITS with Dexa: 1% Insulin-Transferrin-Selenium (ITS+™) and 100 nM dexamethasone (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany),
3) ITS with Dexa + IGF-1 + TGF-β1: 1% Insulin-Transferrin-Selenium (ITS+™), 100 nM dexamethasone, 50 ng mL⁻¹ insulin-like growth factor (IGF)-1 (R&D Systems, Minneapolis, MN, USA) and 50 ng mL⁻¹ transforming growth factor (TGF)-β1 (Peprotec, Hamburg, Germany),
4) FBS: 10% fetal bovine serum (FBS, Pan Biotech, Aidenbach, Germany).

1.2 Immunocytochemistry

After three days of cultivation in the different media compositions, the chondrocytes were washed once with phosphate-buffered saline (PBS; Biochrom GmbH, Berlin, Germany) and fixed for 10 min at room temperature (RT) with 4% paraformaldehyde (ROTI ® Histofix, Carl Roth GmbH + Co. KG, Karlsruhe, Germany). After fixation, cells were washed again and permeabilized with 0.2% Triton-X100 (Merck, Darmstadt, Germany) for 10 min. For blocking the unspecific binding sites, cell-seeded coverslips were incubated with bovine serum albumin (BSA; Sigma-Aldrich) with a concentration of 5% in PBS for one hour at RT after another washing step with PBS. To stain the primary cilium, cells were incubated with anti-acetylated α-tubulin (6-11B-1) (RRID: AB 628409) labeled with Alexa Fluor 647 (sc-23950 AF647, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:200 in PBS at 4°C overnight. Additionally, the Actin cytoskeleton was stained with Acti-stain 488 Fluorescent Phalloidin (Cytoskeleton, Inc., Denver, CO, USA) diluted 10 in PBS for 30 min at RT. Afterward, cells were washed three times with PBS, and the coverslips were fixed with Fluoroshield™ (Sigma-Aldrich) containing 4’,6-Diamidino-2-phenylindole (DAPI).

1.3 Image acquisition

Three-dimensional fluorescence images of stained cells were acquired with a ZEISS ELYRA LSM 780 confocal laser scanning microscope (CLSM) (Carl Zeiss AG, Oberkochen, Germany). Images were recorded using a Plan-Apochromat 63×/1.40 Oil DIC M27 objective (Carl Zeiss AG, Oberkochen, Germany). The distance of two layers was 0.2814 µm and the resolution 1024 × 1024 pixels (scan magnification: 0.6, pixel length: 0.2196 µm).