Icyt synergy sy3200 cell sorter

PBMCs were stained with fluorochrome-conjugated antibodies at a density of 2×10⁴ cells per 1 uL of 1× PBS for 30 min on ice. The following fluorochrome-conjugated antibodies were used for flow cytometry antibody staining: FITC anti-human CD8a (clone: RPA-T8), APC anti-human CD69 (clone: FN50), APC/Cy7 anti-human CD4 (clone: RPA-T4), Pacific Blue anti-human CD3 (clone: UCHT1) and PE anti-human HLA-DRB1 (clone: NFLD. D2). All fluorochrome-conjugated antibodies were purchased from BioLegend. PBMCs were fixed with 500 uL of Biolegend Fixation Buffer per 10⁶ cells for 20 min at ambient temperature. The iCyt Synergy SY3200 Cell Sorter (Sony Biotechnology Inc.) was used in conjunction with WinList 9.0.1 software (Verity Software House) for flow cytometry sample processing and analysis, respectively. PBMCs were gated to select for CD3+ CD8+ HLA-DBR1+ T cells (online supplementary figure 1). The median fluorescence intensity (MFI) of PE anti-human HLA-DRB1 was measured for the CD3+ CD8+ T cell population and normalised to the background PE anti-human HLA-DRB1 MFI of the unstained PBMC population. The normalised MFI (nMFI) was determined by the following equation: $nMFI=\frac{PEMF{I}_{sample}}{PEMF{I}_{unstainedcontrol}}$ .^{26 (link)} FlowJo version 10 was used to generate MFI histograms (FlowJo LLC).

Peripheral blood mononuclear cells (PBMCs) isolated from 12 healthy individuals were initially collected by density gradient centrifugation and immediately stored in liquid nitrogen. Cells were thawed, treated with 25 U/mL benzonase, and incubated at 37°C in RPMI/10% heat inactivated fetal bovine serum for 90 minutes. Thawed PBMCs had a minimum viability of 90%, with an average viability of 98.1% ± 3.6%, measured by tryphan blue staining. Primary monocytes were then isolated from thawed peripheral blood mononuclear cells via negative selection using the Pan Monocyte Isolation Kit, following the manufacturer’s instructions (Miltenyi Biotec Inc., San Diego California). The remaining monocyte-depleted PBMCs were flushed from the magnetic column, and DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen, Hilden Germany). RNA was isolated from primary monocytes using the Direct-zol RNA Isolation Kit (Zymo Research, Irvine California), and then DNase treated using the TURBO DNA-free kit (Invitrogen, Carlsbad California). The purity of the isolated monocytes was measured by flow cytometry using the iCyt Synergy SY3200 cell sorter (Sony Biotechnology, Inc, San Jose California), staining with APC/Cy7 anti-CD14 (BioLegend, San Diego California). Monocyte purity was found to be greater than 90%.