Histopaque histopaque 1077 cell separation medium

Venous blood samples were collected in heparin tubes, and PBMCs were isolated using Ficoll gradient centrifugation, as previously described in^{64 (link)}. Whole blood was transferred to conical tubes and centrifuged at 2000 × g for 5 min. Plasma samples were then collected, and the remaining blood was diluted with RPMI (Corning, Manassas, VA, USA). Diluted blood was then layered over an appropriate volume of Histopaque (Histopaque-1077 Cell Separation Medium, Sigma Aldrich, St. Louis, MO, USA) and centrifuged for 20 min at 500 × g at room temperature without brake. The PBMC layer was carefully removed, transferred to a fresh tube, and washed with RPMI. Cells were stained and counted using Automated Cell Counter (BioRad, Hercules, California, USA). Isolated PBMCs were then cryopreserved in a freezing medium containing 10% dimethyl sulfoxide (DMSO) (MP Biomedicals, LLC, Irvine, CA, USA) and 90% heat-inactivated fetal bovine serum (FBS) and stored in liquid nitrogen for further use.

Whole blood from all participants was collected in heparin-coated tubes and stored at room temperature prior to processing. In brief, PBMCs were isolated by density gradient sedimentation. Whole blood was transferred to conical tubes and then centrifuged at 2000 × g, at room temperature (RT) for 5−10 min. Plasma was then collected, aliquoted and stored at −80 °C for further use. The remaining blood was diluted with RPMI (Corning, Manassas, VA, USA), layered over an appropriate volume of room temperature Histopaque (Histopaque-1077 Cell Separation Medium, Sigma Aldrich, St. Louis, MO, USA), and then centrifuged for 20 min at 448 × g at room temperature without brake. After centrifugation, the PBMC layer was carefully collected, transferred to a conical tube and washed with RPMI. An aliquot of cells was stained with trypan blue and counted using Automated Cell Counter (BioRad, Hercules, California, USA). Isolated PBMCs were then cryopreserved in a cryovial in cell recovery freezing medium containing 10% dimethyl sulfoxide (DMSO) (MP Biomedicals, LLC, Irvine, CA, USA) and 90% heat-inactivated fetal bovine serum (FBS) and stored at −80 °C in a Mr. Frosty freezing container overnight before being transferred to liquid nitrogen for further storage.

Whole blood from all participants was collected in heparin-coated tubes and stored at room temperature prior to processing. In brief, PBMCs were isolated by density gradient sedimentation. Whole blood was transferred to conical tubes and then centrifuged at 2000 rpm, at room temperature (RT) for 5–10 minutes. Plasma was then collected, aliquoted and stored at −80°C for further use. The remaining blood was diluted with RPMI (Corning, Manassas, VA, USA), layered over an appropriate volume of room temperature Histopaque (Histopaque-1077 Cell Separation Medium, Sigma-Aldrich, St. Louis, MO, USA), and then centrifuged for 20 min at 2000 rpm at room temperature without brake. After centrifugation, the PBMC layer was carefully collected, transferred to a conical tube and washed with RPMI. An aliquot of cells was stained with trypan blue and counted using Automated Cell Counter (Bio-Rad, Hercules, California, USA). Isolated PBMCs were then cryopreserved in a cryovial in cell recovery freezing medium containing 10% Dimethyl Sulfoxide (DMSO) (MP Biomedicals, LLC, Irvine, CA, USA) and 90% heat inactivated fetal bovine serum (FBS) and stored at −80°C in a Mr. Frosty freezing container overnight before being transferred to liquid nitrogen for further storage.