Robosep cell separationsystem

To perform prospective bioenergetic assessments of the clonal CD138+
plasma cells from the bone marrow, freshly obtained bone marrow aspirates from
patients underwent Ficoll-Paque gradient separation for plasma processing which
was stored for later analysis at – 80 °C. The remnant cellular
component of the bone marrow aspirate underwent red cell lysis using ACK lysis
buffer. The clonal plasma cells were extracted using positive selection by
mixing the cells with a CD138 positive selection cocktail and anti-CD138
magnetic-activated cell separation microbeads (ROBOSEP™ cell separation
system, StemCell Technologies Inc.) in an automated RoboSep cell separation
system. The purity of the sorted clonal plasma cells was confirmed via light
chain restriction using the slide-based immunofluorescent method.

Spleens from 4get mice were isolated at 8-10 weeks of age and dissociated into single cell suspensions and RBC were lysed using red cell lysing buffer (Sigma-Aldrich), washed, filtered and counted. Single cell preparations were stained with a custom cocktail of biotin-labeled Abs containing anti-CD8α, CD11b, CD19, CD24, CD62L, B220, F4/80, Gr-1, and Ter119. Labeled cells were loaded onto a RoboSep cell separation system using a custom enrichment reagent kit and protocol according to the manufacturer's instructions (Stemcell Technologies, Vancouver BC). iNKT cells were enriched up to 20% and eGFP bright cells were then sorted by FACSAria II (BD Biosciences). Sorted cells were 99% GFP⁺ with the purity of the cells collected being typically >91% iNKT cells, as assessed using CD1d tetramers loaded with αGalCer (Supplementary Fig. 1).