Researchers explain how lipids can control immune response kingscollegelon NatureComms
For in vivo models of disease, WT mice were intraperitoneally injected with 200 μl of clodronate liposomes and 3 days later were adoptively transferred with 3 × 10WT or CD1d-KO BMDCs. Mice were injected i.p. with 40 mg/kg of SSO or vehicle control 4 h prior to LPS orinjection. For induction of LPS-induced inflammation, mice were injected i.p. with 5 μg/g of LPS or vehicle control. Mice were bled via tail prick immediately prior to LPS injection and 4 h post injection.
For lipid raft staining, pMacs were plated on glass cover slips and left to adhere for 2 h. Cells were washed once with PBS, and lipid rafts stained according to manufacturer’s instructions using the Vybrant Lipid Raft Labeling Kit . Briefly, cells were incubated for 10 min with Alexa488-conjugated cholera toxin subunit B at 4 °C, followed by cross-linking with an anti-CTB antibody for 15 min at 4 °C. Cells were washed twice in cold PBS and fixed with 4% paraformaldehyde for 15 min.
Proximity ligation assays were performed using DuoLink In Situ Detection Reagents Orange according to manufacturer’s recommendations using primary mouse anti-CD1d and rabbit anti-CD36 antibodies . Images were acquired using an inverted Zeiss LSM 710 microscope. At least 6 different images per experiment were analysed within Fiji .RNA was extracted from pMacs isolated from 4 WT and 4 CD1d-KO mice using the RNAeasy micro kit following the manufacturer’s instructions. RNA sequencing was performed as described. Briefly, libraries were created using a polyA KAPA mRNA HyperPrep kit .
The raw counts were then imported into R/Bioconductor . DESeq2 was used to estimate different size factors between samples, and a model was used to find genes that were differentially expressed between genotypes. Gene ontology analysis were performed using PANTHER analysis tools
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