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Detection of accessible cholesterol in primary cilia using purified His-ALOD4-mNeon in 3T3 Fibroblasts
Output Details
Description
There exist at least three different pools of cholesterol in the plasma membrane: the essential pool, the sphingomyelin-sequestered pool, and the accessible pool (Radhakrishnan et al, 2020). Ciliary Hedgehog signaling is regulated by the accessible pool of cholesterol (Kinnebrew et al, 2019), and His-mNeon-FLAG-ALOD4, a toxin-based probe, can be used to visualize this accessible pool. Here, we present a method for staining and measuring the amount of accessible cholesterol on cilia in 3T3 fibroblasts stably expressing Somatostatin Receptor (SSTR3)-mApple, a ciliary marker. The protocol described here is based upon previously established methods (Kinnebrew et al., 2019; Johnson and Radhakrishnan, 2021) and has also been used successfully to label cilia in RPE cells.
Identifier (DOI)
10.17504/protocols.io.rm7vzx2qxgx1/v1