Human plasma was isolated from blood samples collected by coagulation promoting. Plasma RTN3 levels were tested by self‐produced RTN3 ELISA embedding assay. Eight microliters of plasma and 92 μL embedding buffer (2.93 g NaHCO2 and 1.59 g Na2CO2 dissolved in ultrapure water) were embedded in 96‐well plates, washed, blocked, and incubated with primary antibody and secondary antibody, and ultimately, 3,3′,5,5′‐tetramethylbenzidine substrate (Solarbio) and stop buffer were added to measure in 450 nm absorbance.
S1P was tested in mouse plasma isolated from blood sampled by eyeball extirpating, in the aortic tissue homogenate of mice dissolved in PBS, and in the supernatant from cells cultured in complete medium for 48 h using S1P ELISA assay kits (Mlbio, Shanghai, China). A NO assay kit (Beyotime) was used to detect the NO content in the culture supernatant. The cell number was counted by a cell counter. Cells were collected, dissolved in PBS, and lysed by freeze–thaw cycles. The lysates were used to detect ceramide and cGMP using ceramide and cGMP ELISA assay kits (Mlbio). Details are provided in the Supplementary Materials and Methods.
S1P was tested in mouse plasma isolated from blood sampled by eyeball extirpating, in the aortic tissue homogenate of mice dissolved in PBS, and in the supernatant from cells cultured in complete medium for 48 h using S1P ELISA assay kits (Mlbio, Shanghai, China). A NO assay kit (Beyotime) was used to detect the NO content in the culture supernatant. The cell number was counted by a cell counter. Cells were collected, dissolved in PBS, and lysed by freeze–thaw cycles. The lysates were used to detect ceramide and cGMP using ceramide and cGMP ELISA assay kits (Mlbio). Details are provided in the Supplementary Materials and Methods.