Experiments were performed with NP cells from non-degenerate IVDs and degenerate IVDs (NP and annulus fibrosus (AF) cells) within passage 1 to 2. Twenty thousand cells were seeded in 8-well chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System) for immunocytochemistry experiments following treatment. Three hundred thousand cells were seeded in 6-well plates (Sarstedt, TC plate 6-well, Cell+, F) for ELISA and RNA extraction following treatment. All cells were left to adhere for 12 to 24 h and then serum-starved in DMEM with 1X insulin-transferrin selenium (ITS, Thermo Fisher, Waltham, MA, USA) for 6 h prior to treatment. To examine the effects of different treatments, healthy cells were treated with either 100 ng/ml Pam2CSK4 (TLR-2/6 agonist, Invivogen), 100 ng/mL Pam3CSK4 (TLR-1/2 agonist, Invivogen), or 5 μg/mL lipopolysaccharide (LPS) (TLR-4 agonist, Invivogen) for 6, 12, 24, and 48 h. Cells were either left untreated (negative control) or treated with 100 ng/mL of Pam2CSK4 for 48 h of which treatment with 100 μM o-vanillin (Sigma-Aldrich, Oakville, ON, Canada) was initiated in the last 6 h of incubation [18, 23, 30].

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.