Chemotaxis assays were conducted similarly to those previously described (56). Briefly, mouse leukocytes, obtained by passing cells isolated from the spleen and subjected to erythrocyte lysis through a 70-μm filter, were suspended in RPMI 1640 medium containing 5% FBS. For the assay, 1 × 106 cells in 100 μl of medium were added to the top chamber of a 6.5-mm-diameter, 5-μm-pore polycarbonate Transwell insert (Costar) and incubated in duplicate with the indicated concentrations of ligand suspended in 600 μl of the medium in the bottom chamber for 2 hours at 37°C. Cells that migrated to the bottom chamber were resuspended, washed, stained with a Live/Dead marker (Aqua Dead, Thermo Fisher Scientific) and antibodies to cell surface markers (CD3, CD4, CD8, CD44, and CD45), fixed with paraformaldehyde, and subjected to cell counting flow cytometric analysis with a BD LSRII flow cytometer. Flow cytometry was performed in the Duke Human Vaccine Institute Research Flow Cytometry Facility (Durham, NC). We were unable to identify a reliable anti-murine CXCR3 antibody for flow cytometry suitable for surface staining. CountBright beads (Thermo Fisher Scientific) were added immediately after bottom chamber resuspension to correct for differences in final volume and any sample loss during wash steps. A 1:10 dilution of input cells was similarly analyzed. Migration was calculated by dividing the number of migrated cells by the number of input cells. In some studies, cells were preincubated for 1 hour at 37°C with PTX (100 ng/ml; List Biological Laboratories), 100 μM LY294002 (Sigma-Aldrich) to inhibit PI3K, or 100 nM AZD5363 (Axon Medchem) to inhibit Akt. Human peripheral blood leukocytes were obtained by venipuncture in accordance with the Duke Institutional Review Board, subjected to erythrocyte lysis, and assayed as described earlier, with the addition of an anti-human CXCR3 antibody. For some samples, reliable anti-human CXCR3 staining was not obtained, which resulted in two fewer replicates in the CD8+CXCR3+ group compared to the CD8+CD44+ group. Analysis was conducted with FlowJo (Ashland, OR) version 10 software. A representative gating tree is shown in fig. S11.

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



Q&A
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.