This dataset is an addition of https://doi.org/10.5281/zenodo.7994589. Here, we have focused on the influence of the magnifications of microscope objectives and image resolution on the results of automated cilia length measurements. 1. Methods 1.1 Cell culture 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, the following supplements were added: 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). 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). To find optimal microscopy parameters for automated detection and length measurement of primary cilia, images were recorded using a Plan-Apochromat 63×/1.40 Oil DIC M27 objective (Carl Zeiss AG, Oberkochen, Germany) or an α Plan-Apochromat 100 × /1.46 Oil DIC M27 Elyra objective (Carl Zeiss AG, Oberkochen, Germany) as well as the following resolutions: 1024 × 1024, 2048 × 2048 and 4096 × 4096 pixels resulting in different voxel sizes (see metadata of files).