Lymphocyte separation media

Human primary trophoblast cells were isolated from first trimester
elective terminations as previously described [24 (link)]. A signed written consent form was obtained from the patients.
The use of placental tissues, specimens and consent forms was approved by the
Yale University Human Investigation Committee (#2000021607). The tissue specimen
was collected in cold, sterile phosphate-buffered saline (PBS) and immediately
transported to the laboratory for cell culture preparation. Briefly, first
trimester placental villous tissues were cut and digested in PBS supplemented
with 0.25% Trypsin (Gibco, Grand Island, NY, USA) for 10 min at 37℃ with
gentle agitation. An equal volume of 10% FBS (Gibco, Grand Island, NY, USA) and
Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA)
was added to inactivate the Trypsin. The supernatant was collected and
centrifuged at 1500 rpm at room temperature for 10 min. The pellet was
resuspended in 5 ml DMEM media supplemented with 10% FBS. This suspension was
laid over lymphocyte separation media (ICN Biomedicals, Inc., Aurora, OH, USA)
and centrifuged at 2000 rpm for 20 min. The interface containing the trophoblast
cells was collected and centrifuged at 1500 rpm for 10 min. Cells were
resuspended in DMEM with 10% FBS and then plated on a 6-well plate to grow.

CD20 mean fluorescence intensity (MFI) values were obtained before and on ibrutinib using peripheral blood mononuclear cells (PBMCs) prepared by density-gradient centrifugation (Lymphocyte Separation Media; ICN Biomedicals, Irvine, CA) and viably frozen in 90% fetal bovine serum (FBS), 10% dimethyl sulfoxide (Sigma, St. Louis, MO) in liquid nitrogen. Enumeration of anti-CD20 binding sites, antibody binding capacity (ABC), was performed on fresh PBMCs and fresh bone marrow cell suspensions in the clinical laboratory using the QuantiBrite system (BD Biosciences, Franklin Lakes, NJ) as previously described (25 ). Cells were analyzed on a FACS Canto II flow cytometer using FACS-DIVA 6.1.1, FCSExpress 4 (BD Biosciences) and FlowJo 10 software (TreeStar, Ashland, OR). In select experiments, the role of NF-κB on CD20 expression was measured using the NF-κB inhibitor Bay 11-7028 (Santa Cruz Biotechnology, Dallas, TX).