CD8+ T cells from blood of H. pylori–infected donors or WT H. pylori–infected mice (9 weeks p.i.) were sorted using a fluorescence-activated cell sorter (FACS) (FACSAria II, BD Biosciences). AGS cells were pretreated with MMP-10 siRNA, NC siRNA (both at 40 nM), or Lipo3000 only (Mock) for 24 hours and then stimulated with WT H. pylori or ΔcagA (MOI = 100) for 24 hours. The culture supernatants were collected and used as source of chemoattractants in a human CD8+ T cell chemotaxis assay. In another set of experiments, mouse primary gastric epithelial cells were purified from gastric tissue single-cell suspensions of uninfected mice with a MACS column purification system using anti-mouse CD326 magnetic beads (Miltenyi Biotec) and then stimulated with WT H. pylori or ΔcagA (MOI = 100) for 24 hours. The culture supernatants were collected, as mentioned above. These culture supernatants were then used as chemoattractants in a mouse CD8+ T cell chemotaxis assay.

In a chemotaxis assay, sorted CD8+ T cells (2 × 105) were transferred into the upper chambers of transwells (5-μm pore). CXCL16 (100 ng/ml) and culture supernatants from various cultures were placed in the lower chambers. After 6-hour culture, migration was quantified by counting cells in the lower chamber and cells adhering to the bottom of the membrane. In some cases, blocking Ab for CXCL16 (20 μg/ml) or corresponding control immunoglobulin G2a (IgG2a; 20 μg/ml) was added into culture supernatants before chemotaxis assay.

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.