Qubit dna broad range or rna broad range fluorescence assays

Viably cryopreserved peripheral blood mononuclear cells (PBMCs) specimens (4–6 million cells) were thawed using AIM-V Serum Free Medium (Thermo Fisher) supplemented with 2% DNase, washed, and resuspended in buffer consisting of PBS, 3% BSA, and 1 mM EDTA. An aliquot of all PBMCs were stained and quantified using the Countess Automated Cell Counter (Life technologies). PBMCs were used to negatively select for monocytes (CD14+) by magnetic bead separation (EasySep Human Monocyte Enrichment Kit without CD16 depletion) or T cells (EasySep Human CD8+ T Cell Enrichment Kit) according to manufacturer’s instructions (StemCell Technologies). DNA and RNA were isolated from enriched monocytes or T cells using the AllPrep DNA/RNA kit (Qiagen) according to the manufacturer’s recommendations for cells. Nucleic acid concentrations were determined using the Qubit DNA Broad Range or RNA Broad Range fluorescence assays (Life Technologies) and Qubit Instrument (Life Technologies).

Viably cryopreserved peripheral blood mononuclear cells (PBMC) were stored in liquid nitrogen and thawed in RPMI 1640 medium (Hyclone, Logan, Utah, USA) containing 10% heat-inactivated fetal-bovine serum (Hyclone) following established protocols[9 (link)]. Cells were stained with propidium iodide (Life Technologies), anti-CD16 Brilliant Violet 421 (Clone 3G8), anti-CD14 BV605 (Clone M5E2), anti-CD3 BV711 (Clone OKT3), anti-CD4 FITC (Clone OKT4), anti-CD7 PE (Clone 6B7), anti-CD11b PerCp/PerCPCY5.5 (Clone ICRF44), anti-CD19 PE-Cy7 (Clone SJ25C1), anti-CD20 PE-Cy7 (Clone 2H7), anti-HLA-DR APC (Clone G46-6). The gating strategy for identification of total monocytes and CD4+ T cells was according to previous reports¹¹. Briefly, monocytes were identified by excluding dead cells, lymphocytes (CD3+), and B Cells (CD19+ or CD20+). Monocytes were isolated as HLA-DR+CD11b+ and CD14+ and CD16+ expression. CD4⁺ T lymphocytes were isolated from the CD3+, CD7+, and CD4+ population of cells. DNA and RNA were isolated from sorted total monocytes and CD4⁺ T lymphocytes using the AllPrep DNA/RNA kit (Qiagen) according to the manufacturer’s recommendations for cells. Nucleic acid concentrations were determined using the Qubit DNA Broad Range or RNA Broad Range fluorescence assays (Life Technologies) and Qubit Instrument (Life Technologies).