Dynabeads flowcomp mouse pan t cd90.2 kit

MRL/Lpr mice splenic T cells were purified with Dynabeads FlowComp Mouse Pan T (CD90.2) Kit and dissociated from beads according to manufacturer's instructions (Thermo Fisher). After staining purified T cells with PE anti-CD138 antibody, TCRβ+CD138− and TCRβ+CD138+ cells were further separated with anti-PE magnetic microbeads (Miltenyi Biotec). Depending on the experimental objective, purified TCRβ+CD138+ and TCRβ+CD138− cells were stimulated with 1 μg/ml anti-CD3 and anti-CD28 antibodies (BD Pharmingen), 10 ng/ml PMA and 100 ng/ml Ion, 100 mg/ml collagenase I, 100 mg/ml collagenase D, 2.5 mg/ml trypsin, 10 μM leupeptin, 500 μM aminophenyl mercuric acetate (all from Sigma–Aldrich), 10 μM focal adhesion kinase inhibitor 14 (Cayman chemicals), 10 μg/ml MMP9, TrypLE, or defined trypsin inhibitor (all from Thermo Fisher) for indicated duration, and the expression levels of cell surface CD138 were quantified by fluorescence-activated cell sorting (FACS). trypsin gene expression levels were quantified by quantitative PCR, and trypsin protein was analyzed by Western blot using rabbit polyclonal trypsin antibody (Thermo Fisher).

For generation of mixed BM chimeras, BM cells were isolated from WT and FL (Foxp3^Cre–ERT2CXCR4^WT/WT or Foxp3^Cre–ERT2CXCR4^FL/FL, respectively) mice, which were maintained on a tamoxifen diet for 4–5 wk, and from CD45.1 mice (as described above). BM cells from WT and FL mice were T cell–depleted using Dynabeads FlowComp Mouse Pan T (CD90.2) Kit (Thermo Fisher Scientific), mixed with similarly prepared competitor (CD45.1) BM cells at a 1:1 ratio, and transplanted (1 × 10⁶ cells total) into lethally irradiated (950 rad) CD45.1 mice by i.v. injection.

Splenic T cells from MRL/Lpr mice were purified with Dynabeads™ FlowComp™ Mouse Pan T (CD90.2) Kit and dissocated from beads as per manufacture's instructions (Thermo Fisher). Purified T cells were staind with PE-conjugated anti-CD138 antibody, and TCRβ+CD138+ and TCRβ+CD138- cells were further separated with anti-PE magnetic MicroBeads (Miltenyi Biotec, Auburn, CA). After three washes with PBS, the purity of isolated TCRβ+CD138+ cells was >95% in all experiments as determined by flow cytometry. For in vivo transfer, purified TCRβ+CD138+ and TCRβ+CD138- cells were suspended in PBS and 1 × 10⁷ cells in 100 μl were i.v. injected into recipient mice. For in vitro culture, CD4+TCRβ+CD138- cells were further isolated from purified TCRβ+CD138- cells using the CD4 (L3T4) MicroBeads (Miltenyi Biotec), and unbound cells were identified as CD8+TCRβ+CD138- cells (over 94% purity). To block mTOR, isolated CD4+TCRβ+CD138- cells were cultured in the presence of 100 nM rapamycin (Tocris Biosciences, Minneapolis, MN). After 3 days of incubation cell viability as well as CD138 and CD4 expression levels were assessed in flow cytometry.