Magnetic ls macs columns

LNCaP cells (ATCC) were a gift from Dr. David Jarrard and were cultured in RPMI medium (Corning) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (HyClone). LNCaP cells were harvested when confluent and frozen in aliquots in growth medium plus 10% DMSO (Fisher Scientific) at -80 ºC and quickly thawed for use in assay validation experiments. White blood cells (WBCs) for assay validation experiments were derived from healthy donor blood or from the blood of a patient with prostate cancer. WBCs were selected on CD45 positivity using magnetic LS MACS columns (Miltenyi). WBCs were frozen in aliquots in PBS plus 10% DMSO (Fisher Scientific) at -80 ºC and quickly thawed for use in assay validation experiments.

Peripheral blood was collected from 19 patients with metastatic NSCLC (S1 Table) with informed written consent under University of Wisconsin IRB approved protocol. A maximum of 50 mL of blood was obtained at any given blood draw using EDTA vacutainers (BD). Whole blood was diluted 1:1 with Hank’s balanced salt solution (HBSS, Lonza) and 30 mL of diluted blood was underlaid with 10 mL of ficoll-paque PLUS (GE Healthcare) per 50 mL conical tube. The blood was centrifuged for 40 min at 974 g, and resulting buffy coats were washed once with HBSS. Peripheral blood mononuclear cells (PBMCs) were incubated with commercially available, pre-conjugated, anti-CD45 magnetic beads (Miltenyi) in a buffer containing 2 mM EDTA (Fisher Scientific) and 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in phosphate buffered saline (PBS, Hyclone), after which CD45⁺ cells were removed using magnetic LS MACS columns (Miltenyi).

Blood was collected and processed as previously described [35 (link), 51 (link)]. Briefly, whole blood collected by venipuncture into EDTA tubes was separated by centrifugation with Ficoll-Paque PLUS (Fisher Scientific). The layer containing mononucleated cells was depleted of CD45+ cells by magnetic LS MACS columns (Miltenyi). CTCs were isolated using an anti-EpCAM goat polyclonal antibody (R&D Systems). RNA was isolated using oligo (dT) Dynabeads (Invitrogen), and DNA was isolated using MagneSil Paramagnetic Particles (PMPs) (Promega) as previously described [34 (link)].