Cd14 pacific blue

Immunophenotyping was performed to determine cell surface marker profiles of hMSCs before and after gene targeting according to ISCT consensus^{28 (link)}. Cells were trypsinized and stained to analyze with flow cytometry, with the following monoclonal antibodies (and clone IDs) from BioLegend Inc. (San Diego, CA): CD105-PE/Cy7 (43A3), CD73-APC (AD2), CD90-PerCP/Cy5.5 (5E10), CD14-Pacific Blue (M5E2), CD19-Pacific Blue (SJ25C1), CD34-Pacific Blue (581), CD45-Pacific Blue (2D1), and HLA-DR-Pacific Blue (L243). All antibodies were used at 1:100 dilutions for staining.

Intracellular cytokine staining was performed on cryopreserved PBMCs of donors randomly selected from each age group (young adults and advanced-age, frail elderly). Briefly, 10⁶ cells (4×10⁶/ml) in X-VIVO 10 media (Lonza, Basel, CH) supplemented with 10% human AB serum (Lonza, Basel, CH) were treated with PBS (mock), 50 ng/ml LPS (Sigma, MO, USA), or 500 ng/ml Pam3CSK4 (Invivogen, CA, USA), and 1x Protein Transport Inhibitor (eBioscience, CA, USA) for 6 hours at 37°C/5% CO₂. Surface staining was performed for 30 min at room temperature with the conjugated antibodies CD14-Pacific Blue (Biolegend, CA, USA), CD16 PE-Cy7, HLA-DR-PerCp Cy5.5 (eBioscience, CA, USA) and CD3-AmCyan (BD Bioscience, ON,CA), and fixed with 1x Fix/lyse buffer (eBioscience, CA, USA) for 10 min. Cells were permeabilized for 30 min with 1x Permeabilization Buffer (eBioscience, CA, USA) at room temperature, and stained with the conjugated antibodies TNF-Alexa Fluor 700, IL-1β-PE, IL-8-APC, and IL-6-FITC (eBioscience, CA, USA) for 30 min at room temperature. Cells were fixed with 2% paraformaldehyde, centrifuged and resuspended in FacsWash prior to analysis. Monocytes were defined as high front scatter (FSC)/Side scatter (SSC), and expressing CD14 and/or CD16 and HLA-DR, but not CD3. Flow cytometry and analysis was performed as described above.