Whole blood was drawn from healthy volunteers obtained in accordance with the recommendations of Canadian Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans and Health Sciences and Affiliated Teaching Hospitals Research Ethics Board (HSREB) with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the HSREB. Primary human monocytes were isolated using RosetteSep™ negative selection human monocyte enrichment cocktail (Stem Cell Technologies, Vancouver, BC, Canada) according to manufacturer’s instructions. Briefly, whole blood was supplemented with respective enrichment cocktails and layered over top of the density gradient Lympholyte-H separation medium (Cedarlane, Ballintemple, Ireland) in SepMate 50-mL conical tubes (StemCell Technologies). Buffy coat was extracted after centrifugation, and cells were washed with PBS + 10 mM EDTA + 2% FBS. Cells were incubated in Gibco RPMI 1640 and supplemented with 10% FBS at 37°C and 5% CO2 and cultured in the presence or absence of IL-30 or IL-27 for the time points described.
Lympholyte h separation medium
Manufactured by Cedarlane
Sourced in Ireland, Canada
Lympholyte-H is a separation medium used to isolate lymphocytes from whole blood. It is a sterile, endotoxin-tested solution designed for density gradient centrifugation.
Automatically generated - may contain errors
2 protocols using lympholyte h separation medium
1
Isolation and Culture of Primary Human Monocytes
2
Isolation and RNA Extraction from PBMCs
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Mononucleated cells were purified from 5 ml of venous peripheral blood from three healthy controls, three patients affected by BMD, and two patients affected by DMD after obtaining their informed consent. Cells were isolated by density gradient centrifugation with Lympholyte-H separation medium (Cedarlane, Burlington, Canada) following manufacturer's instructions.
Total RNA from PBMCs was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol including DNase treatment. Quality and quantity of RNA were determined using a NanoDrop spectrophotometer. The cDNA was synthesized from 400 ng of total RNA with the iScript cDNA Synthesis Kit (Bio-Rad). Each RNA sample was checked for genomic DNA contamination.
Total RNA from PBMCs was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol including DNase treatment. Quality and quantity of RNA were determined using a NanoDrop spectrophotometer. The cDNA was synthesized from 400 ng of total RNA with the iScript cDNA Synthesis Kit (Bio-Rad). Each RNA sample was checked for genomic DNA contamination.
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