Ficoll paque plus method

Human NSCLC cell lines (A549, NCI-H460 and NCI-H1703) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotic-antimycotic solution (Life Technologies, Seoul, Korea). Human hepatocellular carcinoma cell line Huh7 was also purchased from the KCLB. Human bronchial epithelial cell line BEAS-2B was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM medium supplemented with 10% FBS and antibiotic-antimycotic solution (Life Technologies). H9 hESCs were cultured on mouse embryonic fibroblast (MEF) feeder cells as described previously [6 (link), 14 (link)]. Human PBMCs were isolated by the Ficoll-Paque Plus method (GE Healthcare, Seoul, Korea). FO myeloma cells were maintained in DMEM medium supplemented with 10% FBS and antibiotic-antimycotic solution (Life Technologies)

PBMCs were isolated using the Ficoll-Paque Plus Method, which was designed for in vitro isolation of lymphocytes (GE Healthcare Bio-sciences AB). Briefly, the blood sample was transferred into a 50 ml conical tube and PBS was added to achieve a 20ml volume. 15 ml of Ficoll- Paque plus was transferred into a separate 50 ml conical tube using a syringe. The 20 ml of diluted blood was gently layered onto the Ficoll. The sample was spun down at 400xg for 30 minutes at room temperature with no brake. The PBMC layer was then collected at the diluted plasma/ficoll interface. PBS was added to the PBMC to bring the volume up to 20 ml. The sample was then spun down at 200xg for 10 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 1 ml of PBS and counted using a TC20 automatic cell counter from Bio-Rad. 4 ml of PBS was added to the sample and spun down at 200xg for 10 minutes, to remove any contaminating Ficoll and platelets/plasma proteins. For RT-qPCR, The supernatant was discarded and the cells where lysed in 1 ml of trizol and allowed to sit at room temperature for 5 minutes before being transferred to a -80 degrees freezer. For RNAseq, the sample was resuspended in 350ul of RLT plus containing beta-mercapto ethanol, vortexed for 30 seconds and then transferred to a -80 degrees freezer.

PBMCs were isolated using the Ficoll-Paque Plus Method (GE Healthcare Bio-sciences AB) using a process that maintains the viability of B and T lymphocytes to account for relative lymphopenia among A-T versus control participants as previously described^{47 (link)}. Briefly, a 7-mL venous blood sample was transferred into a 50-mL conical tube and PBS was added to achieve a 20-mL volume. 15 mL of Ficoll-Paque Plus was transferred into a separate 50-mL conical tube using a syringe. The 20 mL of diluted blood was gently layered onto the Ficoll. The sample was spun at 400 ×g for 30 minutes at room temperature with no brake. The PBMC layer was then collected at the diluted plasma/Ficoll interface. PBS was added to the PBMCs to bring the volume up to 20 mL. The sample was then spun at 200 ×g for 10 minutes at room temperature. The supernatant was discarded and the cells were resuspended in 1 mL of PBS and counted using a TC20 automatic cell counter (Bio-Rad). 4 mL of PBS was added to the sample and spun at 200 ×g for 10 minutes to remove any contaminating Ficoll, platelets, and plasma proteins. For transcriptional and DNA methylation profiling, the sample was resuspended in 350 µL of RLT plus (Qiagen) containing 1% beta-mercaptoethanol, vortexed for 30 seconds, and then transferred to a −80 °C freezer.