Dynabeads flowcomp human cd14 kit

Monocytes were isolated from the whole blood of two healthy volunteers. First, the erythrocyte lysis step was applied (RBLC reagent, A&A Biotechnology) followed by Dynabeads® FlowComp™ Human CD14 kit (Thermo Fisher Scientific). The CD14 antibody was mixed with the sample and the CD14 + monocytes that bound the specific antibodies were captured by the beads and separated on a magnet. At the final step, the CD14 + monocytes were released from the beads by adding a release buffer. Subsequently, isolated monocytes were seeded on membranes and non-adherent cell culture plates (control group) and cultured for 21 days in DMEM medium supplemented with 10% ultra-low IgG bovine serum and penicillin/streptomycin 100 U/ml each. Culture media were harvested and cytokine presence was detected with a semi-quantitative antibody array consisting of 120 targets (ab193656, Abcam) according to the manufacturer protocol. Array membranes were analyzed with G:Box Chemi XX9 and GeneTools software (Syngene).

CD14⁺ monocytes were isolated from PBMCs (Hemacare) with positive selection using Dynabeads™ FlowComp™ Human CD14 Kit according to manufacturer’s instructions (Thermo Fisher Scientific). Then, monocytes were seeded in six-well plates at a density of 2 × 10⁶ cells per well and cultured at 37 °C/5% CO₂ for 6 days in complete RPMI 1640 medium containing 10% FBS, 100 ng/mL GM-CSF (R&D system), and 40 ng/mL IL-4 (R&D system) to generate monocyte-derived immature DCs (iDCs). To generate v7D- or vOka-pulsed DCs, iDCs were seeded in 6-well plates at a density of 5 × 10⁵ cells per well and incubated with cell-free viruses of v7D or vOka (MOI = 0.01), or mock-infected MRC-5 cell lysate as a negative control, at 37 °C/5% CO₂ over a period of 5 days. LDH release (Thermo Fisher Scientific) and cytokines/chemokine analysis (Millipore, MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel) in the supernatants during co-incubation were performed daily per the manufacturer’s instructions. GM-CSF and IL-4 were maintained in a culture medium during all DC experiments.

Human blood monocytes were isolated with a Dynabeads FlowComp human CD14 kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Briefly, whole blood samples were incubated with FlowComp human antibody to CD14 for 10 min at 4 °C, then centrifuged in isolation buffer for 15 min at 350 g at 4 °C. The pellet was incubated with FlowComp Dynabeads under rolling and tilting for 15 min at 4 °C. Subsequently, the sample was mixed with isolation buffer and placed on a magnet for 3 min. The supernatant containing the CD14⁻ cells was removed. After the washing steps were repeated three times, the bead-bound CD14⁺ cells were resuspended in 1 mL FlowComp release buffer and incubated under rolling and tilting for 10 min at 4 °C. Finally, the beads were washed according to the manufacturer's protocol, and the bead-free CD14⁺ cells were resuspended in the medium and stored at 4 °C until use.
To isolate mouse peritoneal macrophages, the mice were intraperitoneally injected with 2 mL of 3% thioglycolate. 4 days later, the mice were euthanized and intraperitoneally injected with sterile PBS. The abdominal cavity was rinsed with PBS repeatedly with a syringe to collect macrophages.