Easysep human cd8 t cell isolation kit

Blood samples from healthy donors were purchased from a local blood bank. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque (GE Healthcare) density gradient separation. CD8⁺ T cells were negatively selected using an EasySep^TM Human CD8⁺ T cell Isolation kit (STEMCELL Technologies). To activate T cells, a total of 3 million PBMCs or CD8⁺ T cells were treated with immobilized monoclonal antibody (mAb) to CD3 (5μg/mL, Cat# 317304, Biolegend) and soluble anti-CD28 (2μg/mL, Cat# 555725, BD Biosciences) in 1ml of RPMI-1640 media supplemented with 10% fetal bovine serum (FBS). The isotype control antibody used was mouse IgG2a,κ (Cat# 554645) from BD Biosciences. After 48 hrs, PBMCs and the conditioned-media were harvested. Activated PBMCs or CD8⁺ T cells were transferred into 0.4μm Transwell Insert (Corning) and placed into 6-well plates with pre-seeded MDA-MB-231 and MCF7 cells. MDA-MB-231 or MCF7 cells were cocultured with PBMCs alone or the conditioned-media or a combination of both for 24 hrs, and then harvested for further analysis. Control experiments were performed in the same manner except that isotype control antibodies were used.

Buffy coats from de-identified healthy human blood donors were purchased from the Gulf Coast Regional Blood Center (Houston, TX); their acquisition or this purpose was approved by the MD Anderson Institutional Review Board. Peripheral blood mononuclear cells were isolated from buffy coat samples via centrifugation using the Ficoll-Paque method. Human CD8⁺ T cells were enriched from peripheral blood mononuclear cells using an EasySep human CD8⁺ T cell isolation kit (Stemcell Technologies, Vancouver, BC, Canada). These cells were cultured in a mixture comprising 45% RPMI-1640 medium, 45% Click medium (Sigma-Aldrich, St. Louis, MO), and 10% fetal bovine serum, referred to in some experiments as regular medium. Mouse CD8⁺ T cells were enriched from splenocytes using an EasySep mouse CD8⁺ T cell isolation kit (Stemcell Technologies). Mouse T cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and were activated with CD3/CD8 Dynabeads (Thermo Fisher Scientific, Waltham, MA), IL-12 (10 ng/mL), and IL-2 (50 U/mL) recombinant proteins. Supernatants of mouse T cell cultures after 24 h were used as conditioned medium.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats via density gradient separation and either used fresh or cryopreserved in fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO). Tissue samples were processed as described previously^{16 (link),51 (link),52 (link)}. Total CD8⁺ T cells were enriched via negative magnetic separation using an EasySep Human CD8⁺ T Cell Isolation Kit (Stem Cell Technologies) or a MojoSort Human CD8⁺ T Cell Isolation Kit (BioLegend). Total CD3⁺ T cells were enriched via negative magnetic separation using a MojoSort Human CD3⁺ T Cell Isolation Kit (BioLegend). CD8-depleted PBMCs were obtained via negative magnetic separation using CD8 MicroBeads (Miltenyi Biotec). The human NALM-6 cell line (DSMZ) was tested for Mycoplasma (Eurofins Genomics) and transduced with a lentiviral vector encoding secreted luciferase (Lucia⁺/NGFR⁺/NALM-6)^{53 (link)}.