Ficoll paque plus

The mice were euthanized and the spleen was surgically removed and placed in PBS. The single cell solution was obtained by passing the spleen through a 70 µm strainer. Splenocytes were separated by gradient centrifugation with Ficoll-Paque™ PLUS (VWR, Radnor, PA, USA) and red blood cells were removed using red blood cells (RBC) lysis buffer (eBioscience, 00-4333-57). Splenocytes were cryopreserved in FBS with 10% Dimethyl sulfoxide (DMSO) until functional experiments. For activation, splenocytes were co-cultured with Ab1 cells in a ratio 10:1. Staining was performed using IFNγ-PE antibody (BioLegend, 505807) and CD107-PE (LAMP-1) antibody (BioLegend, 121611) together with protein transport inhibition using Golgi Stop (BD Pharm, 554715). T cell proliferation was determined based on the dilution of CellTrace™ carboxyfluorescein succinimidyl ester (CFSE) dye (Thermo Fisher Scientific Corp.) and cytotoxic activity was measured using PrestoBlue™ Cell Viability Reagent (Thermo Fisher Scientific Corp.).

For the initial studies, peripheral blood mononuclear cells (PBMCs) were obtained from apheresis leukoreduction filters using traditional density centrifugation method. The Brigham and Women’s hospital specimen bank provided the samples from consented patients. The cell suspension was diluted with Dulbecco’s phosphate buffered saline (PBS) 1:1 before being layered onto density centrifugation medium (Ficoll-paque plus, VWR, #17-1440-02) and centrifuged for 30 min at 400 g without brake. The PBMCs layer was aspirated and rinsed twice at 200 g for 8 min to remove platelets. PBMCs were then used for subsequent aptamer validation or frozen for system optimization experiments. For magnetic bead studies and system validation, fresh whole blood samples were purchased from Research Blood Components (Boston, MA) and diluted with an equal volume of PBS + 2% fetal calf serum. PBMCs were obtained by following the manufacturer’s instruction with the Sepmate procedure (Stem Cell Technologies, #15460).

Blood was collected from healthy individuals with informed consent (Westmead Hospital Human Research Ethics Committee Approval 1425). Ficoll-Paque Plus (VWR International) was used to isolate peripheral blood mononuclear cells (PBMCs) as previously described (41 ). The generation of LCLs was carried out as previously described (14 (link)). Briefly, fresh or frozen PBMCs were incubated for 1 hr at 37°C with supernatant from the EBV B95-8 cell line, after which the cells were suspended in RPMI-1640 medium (Lonza) containing 10% fetal bovine serum (FBS, Sigma) and 2 mM L-glutamine (Life Technologies). The cells were plated at densities of 5 × 10⁶ cells per well in 48-well plates. The medium was supplemented weekly until the cells were expanded into a 25 cm2 flask. Expanded LCLs were cryopreserved in 10% DMSO (MP Biomedical) 50% FBS and RPMI-1640.

Balb/CByJ mice were euthanized and spleens were surgically removed and placed in PBS. Single-cell solution was obtained by passing an organ through 70-µm strainer. Splenocytes were separated by gradient centrifugation with Ficoll-Paque™ PLUS (VWR). Red blood cells were removed using RBC lysis buffer (eBioscience, 00-4333-57). Splenocytes were cryopreserved in FBS with 10% DMSO until functional experiments. For activation, splenocytes were co-cultured with Ab1 cells at a ratio of 10:1.
Staining was performed using IFNγ-PE antibody (BioLegend, 505807) and CD107-PE (LAMP-1) antibody (BioLegend, 121611) together with protein transport inhibition with Golgi Stop (BD Pharm, 554715). T cell proliferation was determined based on dilution of CellTrace™ CFSE dye (Thermo Fisher). CD4+ activation was analyzed using CD25-PE antibody (BioLegend, 102007) and CD134 (OX40)-BV421 antibody (BioLegend, 119411).