B cells were then selected by negative sorting by using EasySep Human B Cell Enrichment Kit Stemcell Technologies Grenoblee, France).
Baksh et al. reported an easy feasibility of CD146 positive and negative sorting with fluorescence sorting [ 41], which is in accordance with our findings.
Subsequently, CD45.1 donor bone marrow cells were isolated, and T cells were depleted either via biotinylated antibodies against CD3 (Biolegend) and CD90 (Biolegend), followed by negative magnetic sorting with IMag™ streptavidin-coated magnetic beads (BD Biosciences), or using MACS CD3 microbeads (Miltenyi) according to the manufacturer's protocol.
CD146+ MSCs have also been isolated from bone marrow by CD34 negative sorting [ 1].
CD4+ T lymphocytes were then isolated by negative selection with magnetic cell sorting (Miltenyi Biotec), in order to prevent any undesirable activation.
All samples were acquired with a FACScalibur or FACSCanto (Becton Dickinson Immunocytometry Sistems) and analyzed with FlowJo software (Treestar Inc .. B cells were isolated from BM by negative selection with immunomagnetic beads followed by cells sorting.
For the induction of IBD, T cells were isolated from the peripheral lymph nodes and spleen of C57BL/6 mice by negative selection with immunomagnetic beads followed by cells sorting.
Endothelial cells were cultured and purified over a 2- to 3-week period by a series of magnetic immunosortings with Dynabeads (Invitrogen), including a single negative sort using antibodies to the Fc III/II receptor (Abd Serotec) to remove macrophages, followed by at least two positive sorts using antibodies to ICAM-2 (Abd Serotec) to enrich for the endothelial cell population.
Both DAPI-negative/Tomato-positive cells ("positive sort") as well as DAPI-negative/Tomato-negative cells ("negative sort") were sorted directly into RLT buffer (QIAGEN) by fluorescence activated cell sorting (FACS) in the core facility at the University of Michigan.
For sorting of gp41 MPER tetramer-binding B cells, single cell splenic suspensions were obtained from 5 10 naïve BALB/c 8 wk females, pooled, and purified by negative depletion with CD43 MACS beads (Miltenyi Biotechnology).
