Easysep human monocytes enrichment kit w o cd16

For ex vivo stimulation and cytokine production, monocytes were isolated from PBMC (EasySep Human monocytes enrichment kit w/o CD16, StemCell, Vancouver, Canada), according to manufacturer instructions. Purified monocyte fraction was stained with anti-CD14-FITC (BD Bioscience, San Jose, CA, USA) and anti-CD11b-APC-Cy7 (Beckman Coulter, Brea, CA, USA) and purity was evaluated via FC. Purified monocyte fraction was plated in 96-wells flat bottom plate (10⁵ monocytes/well) and incubated for 1 h in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% L-glutamine and 1% sodium-pyruvate at 37 °C. After that, culture media was removed, and cells were washed with warmed PBS as extra of purification. New warmed culture media was combined with 10 ng/mL of LPS and monocytes were incubated overnight. Supernatant was collected and stored at −20 °C.
For RNA extraction, monocytes were isolated from PBMC (CD14 microbeads, MiltenyiBiotec, Bergisch Gladbach, Germany), according to manufacturer instructions. Purified monocytes were stained with anti-CD14-FITC (BD Bioscience, San Jose, CA, USA) and anti-CD11b-APC-Cy7 (BC), and purity was evaluated via FC. Purified monocytes (purity > 90%) were used to extract RNA.

For ex vivo stimulation and cytokine production, monocytes were isolated from PBMC (EasySep Human monocytes enrichment kit w/o CD16, StemCell, Vancouver, BC, Canada), according to the manufacturer’s instructions. The purified monocyte fraction was stained with anti-CD14-FITC (BD Bioscience, San Jose, CA, USA), and purity was evaluated via flow cytometry. Purity of monocytes was >90% in all cases. The purified monocyte fraction was plated in a 96-well flat bottom plate (100,000 monocytes per well) and incubated for 1 h in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% L-glutamine, and 1% sodium-piruvate at 37 °C. After that, culture media were removed, and cells were washed with warmed PBS. New warmed culture media were added with 10 ng/mL of LPS, and monocytes were incubated overnight. Supernatant was then collected and stored at −20 °C.
Frozen supernatant samples were thawed and centrifuged. Eleven cytokines were detected using the MILLIPLEX Map Human Cytokine/Growth Factor panel (Merck KGaA, Darmstadt, Germany): IL6, TNFa, IL10, IL1b, GM-CSF, IL18, IL8, MIP1a, IL1Ra, CCL2, and CXCL10, according to the manufacturer’s instructions. Plates were analyzed by a Luminex 100 platform (One lambda, ThermoFisher Scientific, Waltham, MA. USA).