We collected fresh peripheral blood from OA and RA patients with heparin sodium anticoagulation tubes and obtained peripheral blood mononuclear cells (PBMCs) using Ficoll-Paque gradient centrifugation. PBMCs were stained with fluorochrome-labelled anti-human monoclonal antibodies (Biolegend Inc., San Diego, CA) to CD45 (clone HI30, Cat No. 304014 / 304007 / 304005), CD14 (clone HCD14, Cat No. 325620), CD19 (clone SJ25C1, Cat No. 363006), CD3 (clone OKT3, Cat No. 317343), CD4 (clone RPA-T4, Cat No. 300538), CD8α (clone RPA-T8, Cat No. 301008), and CD16 (clone 3G8, Cat No. 302008) for 15 min at room temperature, followed by DAPI (Cat No. 422801) staining for 10 min. Using a flow cytometer, antibody-stained patient lymphocytes were sorted into monocytes (DAPI-CD45+CD3+CD19-CD14+), B cells (DAPI-CD45+CD3-CD19+), CD4+ T cells (DAPI-CD45+CD3+CD19-CD4+CD8-), and CD8+ T cells (DAPI-CD45+CD3+CD19-CD4+CD8+). At least 50,000 cells were enriched. Post-sort purities of each cell type were ensured for > 95% with flow cytometry. For monocyte subpopulation analysis, monocytes were classified as classical (DAPI-CD45+CD3+CD19-CD14++CD16-), intermediate (DAPI-CD45+CD3+CD19-CD14++CD16+), and non-classical (DAPI-CD45+CD3+CD19- CD14+CD16+).

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.