CRISPR guide RNA (gRNA) was obtained for VPS35, Cav1, and Cltc from Sigma-Aldrich and transfected using protocols from our previous publications (9, 93). After transfection, cells were incubated for 48 hours before treatment. The lentivirus-based CRISPR-Cas9 KO plasmid against gene Vps35, with target site 5′-CAAGTCATTTCCTCAATCCAGG-3′ in a U6gRNA-Cas9-2A-RFP vector, was purchased from Sigma-Aldrich. The lentivirus-based CRISPR-Cas9 plasmids, pLV-U6g-EPCG-Cav1 and pLV-U6g-EPCG-Cltc with the Cav1 and Cltc gRNA target sequences 5′-GTTGAGATGCTTGGGGTCGCGG-3′ and 5′-TACTGAAGCCAATGTTTGCTGG-3′, respectively, were purchased from Sigma-Aldrich. To make the lentivirus, the lenti–CRISPR-Cas9 Vps35 KO plasmid and nontarget control plasmid were transfected into 293FT cells using the MISSION Lentiviral Packaging Mix from Sigma-Aldrich according to the manufacturer’s instructions. The lentivirus was harvested 48 hours after transfection and added to the microglial cell line at a multiplicity of infection of 100 to KD VPS35, caveolin-1, and clathrin expression.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.