Preparation of Cholesterol Crystals

CC were prepared as described elsewhere (Samstad et al. 2014 (link)). Briefly, 100 mg of ultrapure cholesterol (Sigma Aldrich) was dissolved in 50 ml of 1-propanol. The solution was mixed with sterile water (1:1.5) and allowed to rest for 20 min for the crystals to stabilize. All the steps were performed in sterile condition and at room temperature. 1-Propanol was removed by evaporation and CC were resuspended in PBS/0.05% human serum albumin (HSA). CC were further tested for endotoxin contamination using Limulus amebocyte lysate assay (LAL) and LPS levels were found to be below the detection limit (0.01EU/ml). The prepared CC were of size range 1–2 μm and stored at 4 °C.