Laurdan staining was conducted as previously described [46 (
link)]. Briefly, after 6 hours LPS treatment or PBS control, cells were stained with Laurdan (Cayman Chemical Company; Ann Arbor, MI). Briefly, 5 μM/L Laurdan was prepared in serum-free DMEM and incubated with 2×10
6 cells/mL for 30 minutes at 37°C. Cells were subsequently washed and fixed using 4% formaldehyde. To evaluate the effect of cholesterol depletion on lipid raft formation and verify Ct-B staining, cells were pretreated with 10 mM/L
methyl-β-cyclodextrin (Cayman Chemical Company) in DMEM containing 0.1% bovine serum albumin for 3 minutes prior to Laurdan labeling. Laurdan was measured by two-photon microscopy (Olympus
FV 1000, oil immersion, 60x objective) at 400–460 nm and 470–530 nm to calculate generalized polarization (GP)-values [46 (
link)].
Two-photon microscopy images of Laurdan labeled cells were captured as 16-bit/channel TIFF format and were processed using Fiji/ImageJ (NIH). The mean intensities of red and green channels were measured of each whole cell by drawing an oval around the cell. GP-values were obtained by the formula, GP= (
I400–600−
I470–530) ÷ (
I400–460+
I470–530), where
I represents the intensities of each region of interest for the respective channels (35).
Psaltis C.E., Fussner L.A., Yaeger M., Aloor J.J., Reece S.W., Kilburg-Basnyat B.J., Varikuti S., Luo B., Inks M., Sergin S., Schmidt C.A., Neufer P.D., Pennington E.R., Fisher-Wellman K.H., Chowdhury S.M., Fessler M.B., Fenton J.I., Anderson E.J., Shaikh S.R, & Gowdy K.M. (2023). The prohibitin complex regulates macrophage fatty acid composition, plasma membrane packing, and lipid raft-mediated inflammatory signaling. Prostaglandins, leukotrienes, and essential fatty acids, 190, 102540.
